Low Polyunsaturated Fatty Acid Oils and Uses Thereof

ABSTRACT

Provided are microalgal oils having a low polyunsaturated fatty acid profile and derivatives of the oils, including acids, esters, epoxides, hydroxylated acids and esters, urethanes, amides, and polymers thereof. Also provided are compositions comprising the oils and their derivatives and their uses in foodstuffs and in industrial and material applications. The compositions include flexible polyurethane foams prepared from the microalgal oils.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/262,070, filed Apr. 25, 2014, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/816,648,filed Apr. 26, 2013; 61/831,571, filed Jun. 5, 2013; 61/887,278, filedOct. 4, 2013; and 61/942,524, filed Feb. 20, 2014. Each of theseapplications is incorporated herein by reference in its entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“473255-Sequence.txt”, created on Dec. 18, 2015 and containing 342,727bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD

Embodiments of the present invention relate to oils/fats, fuels, foods,and oleochemicals and their production from cultures of geneticallyengineered cells. Specific embodiments relate to oils with a highcontent of triglycerides bearing fatty acyl groups upon the glycerolbackbone in particular regiospecific patterns, highly stable oils, oilswith high levels of oleic or mid-chain fatty acids, and productsproduced from such oils.

BACKGROUND

Polyunsaturated fatty acids (PUFAs) are often present in sizable amountsin high oleic oils and high oleic oil derivatives that are used infoodstuffs and as oleochemicals in industrial and material applications.Many of these oils are obtained from plants and plant seeds, where PUFAsserve as important metabolic compounds necessary for growth andsurvival. The table below shows the approximate fatty acid content ofmajor commercial vegetable oils having a high oleic acid variety.

Oil C18:1 PUFAs Soybean Oil 30% 60% High Oleic Soybean Oil 75% 13%Canola Oil 60% 35% High Oleic Canola Oil 70% 15% Mid Oleic Sunflower Oil65% 25% High Oleic Sunflower Oil 80% 10% Safflower Oil 15% 75% HighOleic Safflower Oil 75% 15%

Soybean, canola, and safflower oils show considerable amounts of PUFAs,and their high oleic versions still show appreciable amounts of PUFAs.Purification of the oils to remove the PUFAs is difficult and costly andthus not feasible on a commercial scale.

Unlike oleic acid which contains a single double bond, PUFAs containmore than one double bond in a fatty acid chain. This increase inunsaturation causes the PUFAs to be susceptible to autoxidation,polymerization, and other chemical reactions. Particularly when exposedto heat, light, and oxidative conditions, the increased reactivity ofthe PUFAs leads to unwanted side products that contaminate the oil. Infoodstuffs such side products can negatively affect taste and smell. Asoleochemicals in industrial and material applications, the side productscan result in a decrease in the stability and desired performancecharacteristics of the oleochemical or material.

SUMMARY OF THE DISCLOSURE

The present invention provides, in one aspect, microalgal oils having alow polyunsaturated fatty acid profile. The fatty acid profile canfurther contain a high oleic or high midchain content. The oils can beused as starting materials for the preparation of derivatives such asbut not limited to acids, esters, epoxides, hydroxylated acids andesters, urethanes, amides, and polymers thereof, including polyesters,polyurethanes, and polyamides. In some aspects, the oils and theirderivatives find use in foodstuffs and in industrial and materialapplications.

In one aspect, the present invention provides a composition comprising amicroalgal oil, wherein the composition is selected from the groupconsisting of a spray oil, a heat transfer fluid, a release agent, and ahydraulic fluid, the microalgal oil having a fatty acid profile of atleast 70% oleic acid and less than 7% polyunsaturated fatty acids.

In some cases, the spray oil is applied to human or animal food. In somecases, the food is fruit, dried fruit, vegetable, dried vegetable, bakedproduct, nuts, candy, or seeds.

In some cases, the release agent optionally comprises one or more of ananti-oxidant, a metal chelator, an oxygen scavenger, an anti-microbialagent, a lubricating additive, or a high viscosity petroleum hydrocarbonoil. In some cases, the release agent is for releasing concrete, food,paper, plastics, or rubber from a mold.

In some cases, the heat transfer fluid optionally comprises one or moreof an anti-oxidant, a metal chelator, an oxygen scavenger, ananti-microbial agent, a pour point depressant, a moisture scavenger, ora colorant. In some cases, the heat transfer fluid is a dielectricfluid. In some cases, the heat transfer fluid is a computer coolantfluid.

In some cases, the hydraulic fluid optionally comprises one or more ofan anti-oxidant, a metal chelator, an anti-corrosion agent, a wearprotection agent, or an anti-foaming agent. In some cases, the hydraulicfluid optionally comprises a phosphate ester. In some cases, thephosphate ester is an alkyl phosphate ester, an aryl phosphate ester, ora mixed alkyl/aryl phosphate ester. In some cases, the phosphate esteris tributyl phosphate, dibutyl phenyl phosphate, tris-iso-propylphenylphosphate, tris-tert-butylphosphate, dioctyl phenyl phosphate, and octyldiphenyl phosphate. In some cases, the hydraulic fluid is an elevatorhydraulic fluid, a construction equipment hydraulic fluid, or a fireresistant hydraulic fluid. In a preferred embodiment, the hydraulicfluid is an elevator hydraulic fluid. In some cases, the hydraulic fluidis used in an aircraft, a steel mill, a die casting machine, ahydroelectric turbine, a hydraulic press, or a compressor.

In any of the foregoing compositions, the fatty acid profile has atleast 75%, 80%, or 85% oleic acid. In some cases, the fatty acid profilehas less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% polyunsaturated fatty acids. Insome cases, the microalgal oil comprises one or more of: at least 10%ergosterol; ergosterol and β-sitosterol, wherein the ratio of ergosterolto β-sitosterol is greater than 25:1; ergosterol and brassicasterol;ergosterol, brassicasterol, and poriferasterol; and the oil isoptionally free from one or more of β-sitosterol, campesterol, andstigmasterol.

In another aspect, the present invention provides a lubricant comprisinga microalgal oil and optionally one or more of an antioxidant, acorrosion inhibitor, a metal deactivator, a lubricity additive, aviscosity index improver, a pour point depressant, an anti-foamingagent, an anti-wear agent, or an extreme pressure resistance additive,and the microalgal oil has a fatty acid profile of at least 70% oleicacid and less than 7% polyunsaturated fatty acids. In some cases, thelubricant is a railroad lubricant, food grade lubricant, gear lubricant,metal working fluid, textile lubricant, combustion engine lubricant, oran electric motor lubricant. In some cases, the microalgal oil of thelubricant has a fatty acid profile of at least 75%, 80%, or 85% oleicacid. In some cases, the fatty acid profile has less than 6%, 5%, 4%,3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%,or 0.01% polyunsaturated fatty acids. In some cases, the microalgal oilcomprises one or more of: at least 10% ergosterol; ergosterol andβ-sitosterol, wherein the ratio of ergosterol to β-sitosterol is greaterthan 25:1; ergosterol and brassicasterol; ergosterol, brassicasterol,and poriferasterol; and the oil is optionally free from one or more ofβ-sitosterol, campesterol, and stigmasterol.

In another aspect, the present invention provides a surfactantcomprising a microalgal oil and optionally one or more of a quaternaryamine, an alkyl polyglycoside, a betaine, a sulfosuccinate, an ethylester sulfonate, or a methyl ester sulfonate, and the microalgal oil hasa fatty acid profile of at least 70% oleic acid and less than 7%polyunsaturated fatty acids. In some cases, the fatty acid profile hasat least 75%, 80%, or 85% oleic acid. In some cases, the fatty acidprofile has less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% polyunsaturated fattyacids. In some cases, the microalgal oil comprises one or more of: atleast 10% ergosterol; ergosterol and β-sitosterol, wherein the ratio ofergosterol to β-sitosterol is greater than 25:1; ergosterol andbrassicasterol; ergosterol, brassicasterol, and poriferasterol; and theoil is optionally free from one or more of β-sitosterol, campesterol,and stigmasterol.

In another aspect, the present invention provides a personal careproduct comprising a microalgal oil, in which the microalgal oil has afatty acid profile of at least 70% oleic acid and less than 7%polyunsaturated fatty acids. In some cases, the fatty acid profile hasat least 75%, 80%, or 85% oleic acid. In some cases, the fatty acidprofile has less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% polyunsaturated fattyacids. In some cases, the microalgal oil comprises one or more of: atleast 10% ergosterol; ergosterol and β-sitosterol, wherein the ratio ofergosterol to β-sitosterol is greater than 25:1; ergosterol andbrassicasterol; ergosterol, brassicasterol, and poriferasterol; and theoil is optionally free from one or more of β-sitosterol, campesterol,and stigmasterol.

In another aspect, the present invention provides a compositioncomprising a derivative of a triacylglyceride, wherein thetriacylglyceride is obtained from a microalgal oil having a fatty acidprofile of at least 70% oleic acid and less than 7% polyunsaturatedfatty acids. In some cases, the microalgal oil comprises one or more of:at least 10% ergosterol; ergosterol and β-sitosterol, wherein the ratioof ergosterol to β-sitosterol is greater than 25:1; ergosterol andbrassicasterol; ergosterol, brassicasterol, and poriferasterol; and theoil is optionally free from one or more of β-sitosterol, campesterol,and stigmasterol.

In some cases, the derivative has a hydroxyl substituted fatty acidmoiety or ester thereof. In some cases, the derivative has a urethanesubstituted fatty acid moiety or ester thereof. In some cases, thederivative is a polyurethane. In some cases, the derivative has anepoxide substituted fatty acid moiety or ester thereof. In some cases,the epoxidized oleic acid is polymerized and the composition is acoating. In some cases, the derivative has an ethoxylated fatty acidmoiety, a sulfonated fatty acid moiety, or a sulfated fatty acid moiety.In some cases, the derivative is a C36 dimer acid, an alkenyl crossmetathesis product, an estolide, azalaic acid, or pelergonic acid. Insome cases, the azelaic acid or pelergonic acid is prepared byperforming ozonolysis of the microalgal oil. In some cases, thederivative is an oleic ester, wherein the ester is a methyl, ethyl,butyl, tert-butyl, propyl, iso-propyl, or an (ethyl)hexyl ester. In somecases, the derivative is an anhydride ester.

In another aspect, the present invention provides a solvent comprisingan oleic acid ester prepared from a microalgal oil having a fatty acidprofile of at least 70% oleic acids and less than 1% polyunsaturatedacids. In some cases, the ester is a methyl, ethyl, butyl, tert-butyl,propyl, iso-propyl, or an (ethyl)hexyl ester. In some cases, themicroalgal oil has a fatty acid profile of at least 75%, 80%, or 85%oleic fatty acids. In some cases, the microalgal oil has less than 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01%polyunsaturated fatty acids. In some cases, the solvent has a VOC(volatile organic compound) as measured by EPA method 24 of less than20%, a Kauri Butanol value as measured by ASTM D1133 of greater than50%, a kinematic viscosity at 40° C. of less than 5 centistokes, and aflash point of greater than 100° C. In some cases, the microalgal oilcomprises one or more of: at least 10% ergosterol; ergosterol andβ-sitosterol, wherein the ratio of ergosterol to β-sitosterol is greaterthan 25:1; ergosterol and brassicasterol; ergosterol, brassicasterol,and poriferasterol; and the oil is optionally free from one or more of(3-sitosterol, campesterol, and stigmasterol.

In another aspect, the present invention provides a cleaning formulationcomprising a solvent as discussed above, and an emulsifier, a non-ionicsurfactant, and water. In some cases, the emulsifier is Videt ME 80. Insome cases, the non-ionic surfactant is Stephan BioSoft N91-8. In somecases, the water is deionized. In some cases, the cleaning formulationfurther comprises limonene, methyl soyate, or methyl ricinoleate. Insome cases, the microalgal oil comprises one or more of: at least 10%ergosterol; ergosterol and (3-sitosterol, wherein the ratio ofergosterol to β-sitosterol is greater than 25:1; ergosterol andbrassicasterol; ergosterol, brassicasterol, and poriferasterol; and theoil is optionally free from one or more of β-sitosterol, campesterol,and stigmasterol.

In another aspect, the solvents provided herein are used as a printingink remover, oil rig and mud wash, industrial parts washer, paint andadhesive remover, and/or as a waterless hand cleaner.

In another aspect, the present invention provides a cross-linkable orcross-linked rubber composition, the composition comprising per onehundred parts by weight of rubber (phr): one or more diene elastomers;from 5 phr to 35 phr of at least one hydrocarbon plasticizing resinhaving a glass transition temperature (Tg) of between 10° C. to 150° C.and an average molecular weight of between 400 g/mol and 2000 g/mol; andfrom 5 phr to 60 phr of an oleaginous microbial oil.

In some cases, the rubber composition further comprises a reinforcingfiller. In some cases, the reinforcing filler comprises an inorganicfiller, carbon black, or a blend thereof. In some cases, the inorganicfiller is silica. In some cases, the reinforcing filler is in an amountfrom 40 to 200 phr.

In some cases, the diene elastomer comprises from 25 to 100 phr of amajority diene elastomer and from 0 to 75 phr of a minority dieneelastomer. In some cases, the majority diene elastomer is selected fromthe group consisting of a copolymer of styrene and butadiene prepared insolution, copolymer of styrene and butadiene prepared in emulsion,copolymer of styrene, and isopropene prepared in solution, terpolymer ofstyrene, butadiene, and isopropene prepared in solution, naturalpolyisoprenes, synthetic polyisoprenes having a cis-1,4 linkage contentgreater than 95%, and mixtures thereof, and the minority diene elastomeris selected from a polybutadiene having a cis-1,4 linkage contentgreater than 90%. In some cases, the majority diene elastomer has a Tgof from −65° C. to −10° C. and the minority diene elastomer has a Tg offrom −110° C. to −75° C. In some cases, the majority diene elastomer isa copolymer of styrene and butadiene prepared in solution. In somecases, the majority diene elastomer has a glass transition temperatureof from −50° C. to −15° C. and greater than 50% trans-1,4 butadienelinkage content. In some cases, the majority diene elastomer is acopolymer of styrene and butadiene prepared in emulsion. In some cases,the majority diene elastomer copolymer has a glass transitiontemperature of from −55° C. to −30° C.

In some cases, the hydrocarbon plasticizing resin has a glass transitiontemperature of from 30° C. to 120° C., a number-average molecular weightof between 400 and 1000 g/mol, and a polymolecularity index less than 2.In some cases, the resin is comprised of units resulting from thepolymerization of a monocyclic or bicyclic unsaturated terpene. In somecases, the resin has a glass transition temperature of from 60° C. to100° C. In some cases, the unsaturated terpene is a monocyclicunsaturated terpene, which in some cases is limonene or dipentene.

In some cases, the resin further comprises one or more units resultingfrom at least one hydrocarbon monomer selected from the group consistingof bicyclic unsaturated terpenes, monocyclic aromatic hydrocarbons,polycyclic aromatic hydrocarbons, cyclic dienes and conjugated dienes.In some cases, the at least one hydrocarbon monomer is selected from thegroup consisting of α-pinene, styrene, alkyl styrene, dicyclopentadieneand isoprene. In some cases, the resin comprises units resulting fromthe polymerization of vinylcyclohexene. In some cases, the resin furthercomprises one or more other units at least one of which has resultedfrom the polymerization of a hydrocarbon selected from the groupconsisting of monocyclic unsaturated terpenes, bicyclic unsaturatedterpenes, monocyclic aromatic hydrocarbons, polycyclic aromatichydrocarbons, cyclic dienes and conjugated dienes.

In some cases, the rubber composition further comprises from 0 phr to 30phr of one or more plasticizing oils extracted from petroleum ofparaffinic, aromatic or naphthenic type. In some cases, the rubbercomposition is devoid of plasticizing oil extracted from petroleum. Insome cases, the rubber composition comprises from 10 phr to 25 phr ofthe hydrocarbon plasticizing resin, and from 15 phr to 30 phr of theplasticizing oil extracted from petroleum.

In some cases, the microbial oil is a microalgal oil comprising C29 andC28 sterols, wherein the amount of C28 sterols is greater than C29sterols.

In some cases, the microbial oil is a microalgal oil comprising one ormore of: at least 10% ergosterol; ergosterol and β-sitosterol, whereinthe ratio of ergosterol to β-sitosterol is greater than 25:1; ergosteroland brassicasterol; ergosterol, brassicasterol, and poriferasterol, andwherein the oil is optionally free from one or more of β-sitosterol,campesterol, and stigmasterol.

In some cases, the microbial oil is a microalgal oil obtained fromheterotrophic oleaginous microalgae. In some cases, the microalgal oilis obtained from microalgae cultivated with sugar from corn, sorghum,sugar cane, sugar beet, or molasses as a carbon source. In some cases,the microalgal oil is obtained from microalgae cultivated on sucrose.

In some cases, the microalgae are Parachlorella, Prototheca, orChlorella. In some cases, the microalgae are Prototheca moriformis.

In some cases, the microalgal oil is obtained from recombinantheterotrophic oleaginous microalgae. In some cases, the microalgal oilhas a fatty acid profile having at least 75%, 80%, or 85% oleic acid. Insome cases, the microalgal oil has a fatty acid profile of at least 50%combined total amount of C10, C12, and C14. In some cases, themicroalgal oil has a fatty acid profile having less than 6%, 5%, 4%, 3%,2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or0.01% polyunsaturated fatty acids.

In another aspect, the present invention provides a tire treadcomprising a rubber composition as discussed above or herein.

In another aspect, the present invention provides a tire comprising atire tread as discussed above.

These and other aspects and embodiments of the invention are describedand/or exemplified in the accompanying drawings, a brief description ofwhich immediately follows, the detailed description of the invention,and in the examples. Any or all of the features discussed above andthroughout the application can be combined in various embodiments of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-14 show fatty acid profiles and melting curves of refined,bleached and deodorized oils from genetically engineered Protothecamoriformis strains, as discussed in Example 4;

FIG. 15 shows the stability of different oils as a function ofantioxidant concentration, as discussed in Example 5;

FIG. 16 shows various properties of natural oils with very low levels ofpolyunsaturated fatty acids in accordance with an embodiment of theinvention; and

FIG. 17 shows a plot of percent solid fat content for various oils asfollows: (a) P. moriformis RBD oil without lipid pathway engineering;(b) Brazilian cocoa butter+25% milk fat; (c) three replicates of P.moriformis RBD oil from a strain expressing hairpin nucleic acids thatreduce levels of a SAD allele thus reducing oleic acid and increasingstearic acid in the TAG profile; (d) P. moriformis RBD oil from a strainoverexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, seeExample 45); (e) Malaysian cocoa butter+25% milk fat; and (f) Malaysiancocoa butter. The cocoa butter and cocoa butter milk fat values areliterature values (Bailey's Industrial Oils and Fat Products, 6^(th)ed.).

FIG. 18 shows the results of thermal stability testing performed onmethylated oil prepared from high-oleic (HO) and high-stabilityhigh-oleic (HSAO) triglyceride oils prepared from heterotrophicallygrown oleaginous microalgae, in comparison to a soya methyl estercontrol sample.

FIG. 19 shows various properties of high-oleic and high-stabilityhigh-oleic algal oils.

FIG. 20 shows TAG composition of strain K-4, strain AU and strain AVoils from flask and fermenter biomass. La=laurate (C12:0), M=myristate(C14:0), P=palmitate (C16:0), Po=palmitoleate (C16:1), S=stearate(C18:0), O=oleate (C18:1), L=linoleate (C18:2), Ln=α-linolenate (C18:3),A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0),Hx=hexacosanoate (C26:0) S—S—S refers to the sum of TAGs in which allthree fatty acids are saturated. In each block of bars, the strains areshown in the order illustrated at the bottom of the figure.

FIG. 21 shows TAG composition of strain AW, strain AX and strain AY oilsfrom shake flask biomass. La=laurate (C12:0), M=myristate (C14:0),P=palmitate (C16:0), Po=palmitoleate (C16:1), S=stearate (C18:0),O=oleate (C18:1), L=linoleate (C18:2), Ln=α-linolenate (C18:3),A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0),Hx=hexacosanoate (C26:0). S—S—S refers to the sum of TAGs in which allthree fatty acids are saturated. In each block of bars, the strains areshown in the order illustrated at the bottom of the figure.

FIG. 22 shows the fatty acid profile and solid fat content of a refined,bleached and deodorized myristate rich oil from a genetically engineeredPrototheca moriformis strain as discussed in Example 52.

FIG. 23 shows the pairwise alignment of heterologous FAE proteinsexpressed in STRAIN Z.

FIG. 24 shows percentage of VOC (volatile organic compounds) as measuredby EPA Method 24. Fatty acid methyl ester prepared from a high oleictriacylglyceride of example 49 was found to have 0.13 g/g (13%) VOC ascompared to 0.95 g/g for limonene and greater than 0.95 g/g for mineralspirits and tolulene.

FIG. 25 shows the density results of the foams of Example 64.

FIG. 26 shows the resilience results of the foams of Example 64.

FIG. 27 shows the tensile strength results of the foams of Example 64.

FIG. 28 shows the elongation results of the foams of Example 64.

FIG. 29 shows the compression force deflection results of the foams ofExample 64.

FIG. 30 shows the compression set results of the foams of Example 64.

FIG. 31 shows the hysteresis loss results of the foams of Example 64.

DETAILED DESCRIPTION I. Definitions

An “allele” is any of one or more alternative forms of a gene whichrelate to one trait or characteristic.

“Antioxidant” refers to a molecule that is capable of inhibiting theoxidation of other molecules. Antioxidants are frequently added toindustrial products. A common use is as stabilizers in fuels andlubricants to prevent oxidation, and in gasolines to prevent thepolymerization that leads to the formation of engine-fouling residues.They are also widely used to prevent the oxidative degradation ofpolymers such as rubbers, plastics and adhesives that causes a loss ofstrength and flexibility in these materials.

“Anti-hydrolysis compound” refers to a molecule that inhibits thedecomposition of a chemical compound by reaction with water.Carbodiimides, for example, can be employed as anti-hydrolysiscompounds. Anti-hydrolysis compounds are commercially available, e.g.,from SpecialChem, among others.

“Anti-wear additive” refers to an additive to a fluid (e.g., alubricating oil) that results in longer machine life due to higher wearand score resistance of the components. Anti-wear additives preventdirect metal-to-metal contact between the machine parts when the oilfilm is broken down. Typically, the additive reacts with the metal onthe part surface and forms a film, which may slide over the frictionsurface. Anti-wear additives typically contain zinc and phosphoruscompounds. Examples of anti-wear additives include zinc dithiophosphate(ZDP), zinc dialkyl dithio phosphate (ZDDP, also acts as a corrosioninhibitor and antioxidant), tricresyl phosphate (TCP, used forhigh-temperature operation), halocarbons (chlorinated paraffins, forextreme pressure operations), glycerol mono-oleate, stearic acid (whichadheres to surfaces via reversible adsorption process under 150° C.,useful for mild contact conditions.

A “natural oil” or “natural fat” shall mean a predominantly triglycerideoil obtained from an organism, where the oil has not undergone blendingwith another natural or synthetic oil, or fractionation so as tosubstantially alter the fatty acid profile of the triglyceride. Inconnection with an oil comprising triglycerides of a particularregiospecificity, the natural oil or natural fat has not been subjectedto interesterification or other synthetic process to obtain thatregiospecific triglyceride profile, rather the regiospecificity isproduced naturally, by a cell or population of cells. In connection witha natural oil or natural fat, and as used generally throughout thepresent disclosure, the terms oil and fat are used interchangeably,except where otherwise noted. Thus, an “oil” or a “fat” can be liquid,solid, or partially solid at room temperature, depending on the makeupof the substance and other conditions. Here, the term “fractionation”means removing material from the oil in a way that changes its fattyacid profile relative to the profile produced by the organism, howeveraccomplished. The terms “natural oil” and “natural fat” encompass suchoils obtained from an organism, where the oil has undergone minimalprocessing, including refining, bleaching and/or degumming, that doesnot substantially change its triglyceride profile. A natural oil canalso be a “noninteresterified natural oil”, which means that the naturaloil has not undergone a process in which fatty acids have beenredistributed in their acyl linkages to glycerol and remain essentiallyin the same configuration as when recovered from the organism.

“Exogenous gene” shall mean a nucleic acid that codes for the expressionof an RNA and/or protein that has been introduced into a cell (e.g. bytransformation/transfection), and is also referred to as a “transgene”.A cell comprising an exogenous gene may be referred to as a recombinantcell, into which additional exogenous gene(s) may be introduced. Theexogenous gene may be from a different species (and so heterologous), orfrom the same species (and so homologous), relative to the cell beingtransformed. Thus, an exogenous gene can include a homologous gene thatoccupies a different location in the genome of the cell or is underdifferent control, relative to the endogenous copy of the gene. Anexogenous gene may be present in more than one copy in the cell. Anexogenous gene may be maintained in a cell as an insertion into thegenome (nuclear or plastid) or as an episomal molecule.

“Fatty acids” shall mean free fatty acids, fatty acid salts, or fattyacyl moieties in a glycerolipid. It will be understood that fatty acylgroups of glycerolipids can be described in terms of the carboxylic acidor anion of a carboxylic acid that is produced when the triglyceride ishydrolyzed or saponified.

“Fixed carbon source” is a molecule(s) containing carbon, typically anorganic molecule that is present at ambient temperature and pressure insolid or liquid form in a culture media that can be utilized by amicroorganism cultured therein. Accordingly, carbon dioxide is not afixed carbon source.

“In operable linkage” is a functional linkage between two nucleic acidsequences, such a control sequence (typically a promoter) and the linkedsequence (typically a sequence that encodes a protein, also called acoding sequence). A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

“Microalgae” are eukaryotic microbial organisms that contain achloroplast or other plastid, and optionally that is capable ofperforming photosynthesis, or a prokaryotic microbial organism capableof performing photosynthesis. Microalgae include obligatephotoautotrophs, which cannot metabolize a fixed carbon source asenergy, as well as heterotrophs, which can live solely off of a fixedcarbon source. Microalgae include unicellular organisms that separatefrom sister cells shortly after cell division, such as Chlamydomonas, aswell as microbes such as, for example, Volvox, which is a simplemulticellular photosynthetic microbe of two distinct cell types.Microalgae include cells such as Chlorella, Dunaliella, and Prototheca.Microalgae also include other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys. Microalgae also include obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate algae species and species of the genusPrototheca.

In connection with a recombinant cell, the term knockdown refers to agene that has been partially suppressed (e.g., by about 1-95%) in termsof the production or activity of a protein encoded by the gene.

Also, in connection with a recombinant cell, the term knockout refers toa gene that has been completely or nearly completely (e.g., >95%)suppressed in terms of the production or activity of a protein encodedby the gene. Knockouts can be prepared by homologous recombination of anoncoding sequence into a coding sequence, gene deletion, mutation orother method.

An “oleaginous” cell is a cell capable of producing at least 20% lipidby dry cell weight, naturally or through recombinant or classical strainimprovement. An “oleaginous microbe” or “oleaginous microorganism” is amicrobe, including a microalga that is oleaginous. An oleaginous cellalso encompasses a cell that has had some or all of its lipid or othercontent removed, and both live and dead cells.

An “ordered oil” or “ordered fat” is one that forms crystals that areprimarily of a given polymorphic structure. For example, an ordered oilor ordered fat can have crystals that are greater than 50%, 60%, 70%,80%, or 90% of the β or β′ polymorphic form.

In connection with a natural oil, a “profile” is the distribution ofparticular species or triglycerides or fatty acyl groups within the oil.A “fatty acid profile” is the distribution of fatty acyl groups in thetriglycerides of the oil without reference to attachment to a glycerolbackbone. Fatty acid profiles are typically determined by conversion toa fatty acid methyl ester (FAME), followed by gas chromatography (GC)analysis with flame ionization detection (FID) as in Example 1. Thefatty acid profile can be expressed as one or more percent of a fattyacid in the total fatty acid signal determined from the area under thecurve for that fatty acid. FAME-GC-FID measurement approximate weightpercentages of the fatty acids. A “sn-2 profile” is the distribution offatty acids found at the sn-2 position of the triacylglycerides in theoil. A “regiospecific profile” is the distribution of triglycerides withreference to the positioning of acyl group attachment to the glycerolbackbone without reference to stereospecificity. In other words, aregiospecific profile describes acyl group attachment at sn-1/3 vs.sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate)and SOP (stearate-oleate-palmitate) are treated identically. A“stereospecific profile” describes the attachment of acyl groups atsn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such asSOP and POS are to be considered equivalent. A “TAG profile” is thedistribution of fatty acids found in the triglycerides with reference toconnection to the glycerol backbone, but without reference to theregiospecific nature of the connections. Thus, in a TAG profile, thepercent of SSO in the oil is the sum of SSO and SOS, while in aregiospecific profile, the percent of SSO is calculated withoutinclusion of SOS species in the oil. In contrast to the weightpercentages of the FAME-GC-FID analysis, triglyceride percentages aretypically given as mole percentages; that is the percent of a given TAGmolecule in a TAG mixture.

The term “percent sequence identity,” in the context of two or moreamino acid or nucleic acid sequences, refers to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection. For sequence comparison todetermine percent nucleotide or amino acid identity, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters. Optimalalignment of sequences for comparison can be conducted using the NCBIBLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. Forexample, to compare two nucleic acid sequences, one may use blastn withthe “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at thefollowing default parameters: Matrix: BLOSUM62; Reward for match: 1;Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties;Gap x drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For apairwise comparison of two amino acid sequences, one may use the “BLAST2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set, forexample, at the following default parameters: Matrix: BLOSUM62; OpenGap: 11 and Extension Gap: 1 penalties; Gap x drop-off 50; Expect: 10;Word Size: 3; Filter: on.

“Recombinant” is a cell, nucleic acid, protein or vector that has beenmodified due to the introduction of an exogenous nucleic acid or thealteration of a native nucleic acid. Thus, e.g., recombinant cells canexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes differently than those genesare expressed by a non-recombinant cell. Recombinant cells can, withoutlimitation, include recombinant nucleic acids that encode for a geneproduct or for suppression elements such as mutations, knockouts,antisense, interfering RNA (RNAi) or dsRNA that reduce the levels ofactive gene product in a cell. A “recombinant nucleic acid” is a nucleicacid originally formed in vitro, in general, by the manipulation ofnucleic acid, e.g., using polymerases, ligases, exonucleases, andendonucleases, using chemical synthesis, or otherwise is in a form notnormally found in nature. Recombinant nucleic acids may be produced, forexample, to place two or more nucleic acids in operable linkage. Thus,an isolated nucleic acid or an expression vector formed in vitro byligating DNA molecules that are not normally joined in nature, are bothconsidered recombinant for the purposes of this invention. Once arecombinant nucleic acid is made and introduced into a host cell ororganism, it may replicate using the in vivo cellular machinery of thehost cell; however, such nucleic acids, once produced recombinantly,although subsequently replicated intracellularly, are still consideredrecombinant for purposes of this invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid.

The terms “triglyceride”, “triacylglyceride” and “TAG” are usedinterchangeably as is known in the art.

II. General

Illustrative embodiments of the present invention feature oleaginouscells that produce altered fatty acid profiles and/or alteredregiospecific distribution of fatty acids in glycerolipids, and productsproduced from the cells. Examples of oleaginous cells include microbialcells having a type II fatty acid biosynthetic pathway, includingplastidic oleaginous cells such as those of oleaginous algae. Specificexamples of cells include heterotrophic or obligate heterotrophicmicroalgae of the phylum Chlorophtya, the class Trebouxiophytae, theorder Chlorellales, or the family Chlorellacae. Examples of oleaginousmicroalgae and methods of cultivation are also provided in Published PCTPatent Applications WO2008/151149, WO2010/06032, WO2011/150410,WO2011/150411, WO2012/061647, and WO2012/106560, including species ofChlorella and Prototheca, a genus comprising obligate heterotrophs. Theoleaginous cells can be, for example, capable of producing 25, 30, 40,50, 60, 70, 80, 85, or about 90% oil by cell weight, ±5%. Optionally,the oils produced can be low in highly unsaturated fatty acids such asDHA or EPA fatty acids. For example, the oils can comprise less than 5%,2%, or 1% DHA and/or EPA. In some cases, the microalgal oils have a higholeic and low polyunsaturated fatty acid profile and derivatives of theoils, including acids, esters, epoxides, hydroxylated acids and esters,urethanes, amides, and polymers can be prepared with the same attributesfor use in foodstuffs and in industrial and material applications. Theabove-mentioned publications also disclose methods for cultivating suchcells and extracting oil, especially from microalgal cells; such methodsare applicable to the cells disclosed herein and incorporated byreference for these teachings. When microalgal cells are used they canbe cultivated autotrophically (unless an obligate heterotroph) or in thedark using a sugar (e.g., glucose, fructose and/or sucrose) In any ofthe embodiments described herein, the cells can be heterotrophic cellscomprising an exogenous invertase gene so as to allow the cells toproduce oil from a sucrose feedstock. Alternately, or in addition, thecells can metabolize xylose from cellulosic feedstocks. For example, thecells can be genetically engineered to express one or more xylosemetabolism genes such as those encoding an active xylose transporter, axylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, axylitol dehydrogenase and a xylose reductase. See WO2012/154626,“GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE”,published Nov. 15, 2012.

The oleaginous cells may, optionally, be cultivated in abioreactor/fermenter. For example, heterotrophic oleaginous microalgalcells can be cultivated on a sugar-containing nutrient broth.Optionally, cultivation can proceed in two stages: a seed stage and alipid-production stage. In the seed stage, the number of cells isincreased from s starter culture. Thus, the seeds stage typicallyincludes a nutrient rich, nitrogen replete, media designed to encouragerapid cell division. After the seeds stage, the cells may be fed sugarunder nutrient-limiting (e.g. nitrogen sparse) conditions so that thesugar will be converted into triglycerides. For example, the rate ofcell division in the lipid-production stage can be decreased by 50%, 80%or more relative to the seed stage. Additionally, variation in the mediabetween the seed stage and the lipid-production stage can induce therecombinant cell to express different lipid-synthesis genes and therebyalter the triglycerides being produced. For example, as discussed below,nitrogen and/or pH sensitive promoters can be placed in front ofendogenous or exogenous genes. This is especially useful when an oil isto be produced in the lipid-production phase that does not supportoptimal growth of the cells in the seed stage. In an example below, acell has a fatty acid desaturase with a pH sensitive promoter so than anoil that is low in linoleic acid is produced in the lipid productionstage while an oil that has adequate linoleic acid for cell division isproduced during the seed stage. The resulting low linoleic oil hasexceptional oxidative stability.

The oleaginous cells express one or more exogenous genes encoding fattyacid biosynthesis enzymes. As a result, some embodiments feature naturaloils that were not obtainable from a non-plant or non-seed oil, or notobtainable at all.

The oleaginous cells produce a storage oil, which is primarilytriacylglyceride and may be stored in storage bodies of the cell. A rawoil may be obtained from the cells by disrupting the cells and isolatingthe oil. The raw oil may comprise sterols produced by the cells.WO2008/151149, WO2010/06032, WO2011/150410, WO2011/1504 WO2012/061647,and WO2012/106560 disclose heterotrophic cultivation and oil isolationtechniques. For example, oil may be obtained by providing orcultivating, drying and pressing the cells. The oils produced may berefined, bleached and deodorized (RBD) as known in the art or asdescribed in WO2010/120939. The raw or RBD oils may be used in a varietyof food, chemical, and industrial products or processes. Even after suchprocessing, the oil may retain a sterol profile characteristic of thesource. Microalgal sterol profiles are disclosed below. See especiallySection XII of this patent application. After recovery of the oil, avaluable residual biomass remains. Uses for the residual biomass includethe production of paper, plastics, absorbents, adsorbents, drillingfluids, as animal feed, for human nutrition, or for fertilizer.

Where a fatty acid profile of a triglyceride (also referred to as a“triacylglyceride” or “TAG”) cell oil is given here, it will beunderstood that this refers to a nonfractionated sample of the storageoil extracted from the cell analyzed under conditions in whichphospholipids have been removed or with an analysis method that issubstantially insensitive to the fatty acids of the phospholipids (e.g.using chromatography and mass spectrometry). The oil may be subjected toan RBD process to remove phospholipids, free fatty acids and odors yethave only minor or negligible changes to the fatty acid profile of thetriglycerides in the oil. Because the cells are oleaginous, in somecases the storage oil will constitute the bulk of all the TAGs in thecell. Examples 1, 2, and 8 below give analytical methods for determiningTAG fatty acid composition and regiospecific structure.

Broadly categorized, certain embodiments provided herein include (i)auxotrophs of particular fatty acids; (ii) cells that produce oilshaving low concentrations of polyunsaturated fatty acids, includingcells that are auxotrophic for unsaturated fatty acids; (iii) cellsproducing oils having high concentrations of particular fatty acids dueto expression of one or more exogenous genes encoding enzymes thattransfer fatty acids to glycerol or a glycerol ester; (iv) cellsproducing regiospecific oils, and (v) genetic constructs or cellsencoding a newly discovered gene encoding an LPAAT enzyme from CupheaPSR23 (see Example 43). The embodiments also encompass the oils made bysuch cells, the residual biomass from such cells after oil extraction,oleochemicals, fuels and food products made from the oils and methods ofcultivating the cells.

In any of the embodiments below, the cells used are optionally cellshaving a type II fatty acid biosynthetic pathway such as microalgalcells including heterotrophic or obligate heterotrophic microalgalcells, including cells classified as Chlorophyta, Trebouxiophyceae,Chlorellales, Chlorellaceae, or Chlorophyceae, or cells engineered tohave a type II fatty acid biosynthetic pathway using the tools ofsynthetic biology (i.e., transplanting the genetic machinery for a typeII fatty acid biosynthesis into an organism lacking such a pathway). Inspecific embodiments, the cell is of the species Prototheca moriformis,Prototheca krugani, Prototheca stagnora or Prototheca zopfii or has a23S rRNA sequence with at least 65, 70, 75, 80, 85, 90 or 95% nucleotideidentity SEQ ID NO: 76. By cultivating in the dark or using an obligateheterotroph, the natural oil produced can be low in chlorophyll or othercolorants. For example, the natural oil can have less than 100, 50, 10,5, 1, 0.0.5 ppm of chlorophyll without substantial purification.

The stable carbon isotope value δ13C is an expression of the ratio of¹³C/¹²C relative to a standard (e.g. PDB, carbonite of fossil skeletonof Belemnite americana from Peedee formation of South Carolina). Thestable carbon isotope value δ13C (‰) of the oils can be related to theδ13C value of the feedstock used. In some embodiments the oils arederived from oleaginous organisms heterotrophically grown on sugarderived from a C4 plant such as corn or sugarcane. In some embodimentsthe δ13C (‰) of the oil is from −10 to −17 ‰ or from −13 to −16 ‰.

In specific embodiments and examples discussed below, one or more fattyacid synthesis genes (e.g., encoding an acyl-ACP thioesterase, aketo-acyl ACP synthase, an LPAAT, a stearoyl ACP desaturase, or othersdescribed herein) is incorporated into a microalga. It has been foundthat for certain microalga, a plant fatty acid synthesis gene product isfunctional in the absence of the corresponding plant acyl carrierprotein (ACP), even when the gene product is an enzyme, such as anacyl-ACP thioesterase, that requires binding of ACP to function. Thus,optionally, the microalgal cells can utilize such genes to make adesired oil without co-expression of the plant ACP gene.

III. Fatty Acid Auxotrophs/Reducing Fatty Acid Levels to GrowthInhibitory Conditions During an Oil Production Phase

In an embodiment, the cell is genetically engineered so that all allelesof a lipid pathway gene are knocked out. Alternately, the amount oractivity of the gene products of the alleles is knocked down so as torequire supplementation with fatty acids. A first transformationconstruct can be generated bearing donor sequences homologous to one ormore of the alleles of the gene. This first transformation construct maybe introduced and selection methods followed to obtain an isolatedstrain characterized by one or more allelic disruptions. Alternatively,a first strain may be created that is engineered to express a selectablemarker from an insertion into a first allele, thereby inactivating thefirst allele. This strain may be used as the host for still furthergenetic engineering to knockout or knockdown the remaining allele(s) ofthe lipid pathway gene. Complementation of the endogenous gene can beachieved through engineered expression of an additional transformationconstruct bearing the endogenous gene whose activity was originallyablated, or through the expression of a suitable heterologous gene. Theexpression of the complementing gene can either be regulatedconstitutively or through regulatable control, thereby allowing fortuning of expression to the desired level so as to permit growth orcreate an auxotrophic condition at will. In an embodiment, a populationof the fatty acid auxotroph cells are used to screen or select forcomplementing genes; e.g., by transformation with particular genecandidates for exogenous fatty acid synthesis enzymes, or a nucleic acidlibrary believed to contain such candidates.

Knockout of all alleles of the desired gene and complementation of theknocked-out gene need not be carried out sequentially. The disruption ofan endogenous gene of interest and its complementation either byconstitutive or inducible expression of a suitable complementing genecan be carried out in several ways. In one method, this can be achievedby co-transformation of suitable constructs, one disrupting the gene ofinterest and the second providing complementation at a suitable,alternative locus. In another method, ablation of the target gene can beeffected through the direct replacement of the target gene by a suitablegene under control of an inducible promoter. In this way, expression ofthe targeted gene is now put under the control of a regulatablepromoter. An additional approach is to replace the endogenous regulatoryelements of a gene with an exogenous, inducible gene expression system.Under such a regime, the gene of interest can now be turned on or offdepending upon the particular needs. A still further method is to createa first strain to express an exogenous gene capable of complementing thegene of interest, then to knockout out or knockdown all alleles of thegene of interest in this first strain. The approach of multiple allelicknockdown or knockout and complementation with exogenous genes may beused to alter the fatty acid profile, regiospecific profile, sn-2profile, or the TAG profile of the engineered cell.

In a specific embodiment, the recombinant cell comprises nucleic acidsoperable to reduce the activity of an endogenous acyl-ACP thioesterase;for example a FatA or FatB acyl-ACP thioesterase having a preference forhydrolyzing fatty acyl-ACP chains of length C18 (e.g., stearate (C18:0)or oleate (C18:1), or C8:0-C16:0 fatty acids. The activity of anendogenous acyl-ACP thioesterase may be reduced by knockout or knockdownapproaches. Knockdown may be achieved, for example, through the use ofone or more RNA hairpin constructs, by promoter hijacking (substitutionof a lower activity or inducible promoter for the native promoter of anendogenous gene), or by a gene knockout combined with introduction of asimilar or identical gene under the control of an inducible promoter.Example 34 describes the engineering of a Prototheca strain in which twoalleles of the endogenous fatty acyl-ACP thioesterase (FATA1) have beenknocked out. The activity of the Prototheca moriformis FATA1 wascomplemented by the expression of an exogenous FatA or FatB acyl-ACPthioesterase. Example 36 details the use of RNA hairpin constructs toreduce the expression of FATA in Prototheca, which resulted in analtered fatty acid profile having less palmitic acid and more oleicacid.

Accordingly, oleaginous cells, including those of organisms with a typeII fatty acid biosynthetic pathway can have knockouts or knockdowns ofacyl-ACP-thioesterase encoding alleles to such a degree as to eliminateor severely limit viability of the cells in the absence of fatty acidsupplementation or genetic complementations. These strains can be usedto select for transformants expressing acyl-ACP-thioesterase transgenes.Alternately, or in addition, the strains can be used to completelytransplant exogenous acyl-ACP-thioesterases to give dramaticallydifferent fatty acid profiles of natural oils produced by such cells.For example, FATA expression can be completely or nearly completelyeliminated and replaced with FATB genes that produce mid-chain fattyacids. In certain specific embodiments, these transformants producenatural oils with more than 50, 60, 70, 80, or 90% caprylic, capric,lauric, myristic, or palmitic acid, or total fatty acids of chain lengthless than 18 carbons. Such cells may require supplementation with longerchain fatty acids such as stearic or oleic acid or switching ofenvironmental conditions between growth permissive and restrictivestates in the case of an inducible promoter regulating a FatA gene.

In an embodiment the oleaginous cells are cultured (e.g., in abioreactor). The cells are fully auxotrophic or partially auxotrophic(i.e., lethality or synthetic sickness) with respect to one or moretypes of fatty acid. The cells are cultured with supplementation of thefatty acid(s) so as to increase the cell number, then allowing the cellsto accumulate oil (e.g. to at least 40% by dry cell weight).Alternatively, the cells comprise a regulatable fatty acid synthesisgene that can be switched in activity based on environmental conditionsand the environmental conditions during a first, cell division, phasefavor production of the fatty acid and the environmental conditionsduring a second, oil accumulation, phase disfavor production of thefatty acid. In the case of an inducible gene, the regulation of theinducible gene can be mediated, without limitation, via environmental pH(for example, by using the AMT3 promoter as described in the Examples).

As a result of applying either of these supplementation or regulationmethods, a cell oil may be obtained from the cell that has low amountsof one or more fatty acids essential for optimal cell propagation.Specific examples of oils that can be obtained include those low instearic, linoleic and/or linolenic acids.

These cells and methods are illustrated in connection with lowpolyunsaturated oils in the section immediately below and in Example 6(fatty acid desaturase auxotroph) in connection with oils low inpolyunsaturated fatty acids and in Example 34 (acyl-ACP thioesteraseauxotroph).

Likewise, fatty acid auxotrophs can be made in other fatty acidsynthesis genes including those encoding a SAD, FAD, KASIII, KASI,KASII, KCS, elongase, GPAT, LPAAT, DGAT or AGPAT or PAP. Theseauxotrophs can also be used to select for complement genes or toeliminate native expression of these genes in favor of desired exogenousgenes in order to alter the fatty acid profile, regiospecific profile,or TAG profile of natural oils produced by oleaginous cells.

Accordingly, in an embodiment of the invention, there is a method forproducing an oil/fat. The method comprises cultivating a recombinantoleaginous cell in a growth phase under a first set of conditions thatis permissive to cell division so as to increase the number of cells dueto the presence of a fatty acid, cultivating the cell in an oilproduction phase under a second set of conditions that is restrictive tocell division but permissive to production of an oil that is depleted inthe fatty acid, and extracting the oil from the cell, wherein the cellhas a mutation or exogenous nucleic acids operable to suppress theactivity of a fatty acid synthesis enzyme, the enzyme optionally being astearoyl-ACP desaturase, delta 12 fatty acid desaturase, or aketoacyl-ACP synthase. The oil produced by the cell can be depleted inthe fatty acid by at least 50, 60, 70, 80, or 90%. The cell can becultivated heterotrophically. The cell can be a microalgal cellcultivated heterotrophically or autotrophically and may produce at least40, 50, 60, 70, 80, or 90% oil by dry cell weight.

IV. Low Polyunsaturated Natural Oils

In an embodiment of the present invention, the natural oil produced bythe cell has very low levels of polyunsaturated fatty acids. As aresult, the natural oil can have improved stability, including oxidativestability. The natural oil can be a liquid or solid at room temperature,or a blend of liquid and solid oils, including the regiospecific orstereospecific oils, high stearate oils, or high mid-chain oilsdescribed infra. Oxidative stability can be measured by the Rancimatmethod using the AOCS Cd 12b-92 standard test at a defined temperature.For example, the OSI (oxidative stability index) test may be run attemperatures between 110° C. and 140° C. The oil is produced bycultivating cells (e.g., any of the plastidic microbial cells mentionedabove or elsewhere herein) that are genetically engineered to reduce theactivity of one or more fatty acid desaturase. For example, the cellsmay be genetically engineered to reduce the activity of one or morefatty acyl Δ12 desaturase(s) responsible for converting oleic acid(18:1) into linoleic acid (18:2) and/or one or more fatty acyl Δ15desaturase(s) responsible for converting linoleic acid (18:2) intolinolenic acid (18:3). Various methods may be used to inhibit thedesaturase including knockout or mutation of one or more alleles of thegene encoding the desaturase in the coding or regulatory regions,inhibition of RNA transcription, or translation of the enzyme, includingRNAi, siRNA, miRNA, dsRNA, antisense, and hairpin RNA techniques. Othertechniques known in the art can also be used including introducing anexogenous gene that produces an inhibitory protein or other substancethat is specific for the desaturase.

In a specific embodiment, fatty acid desaturase (e.g., Δ12 fatty aciddesaturase) activity in the cell is reduced to such a degree that thecell is unable to be cultivated or is difficult to cultivate (e.g., thecell division rate is decreased more than 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 97 or 99%). Achieving such conditions may involve knockout,or effective suppression of the activity of multiple gene copies (e.g.2, 3, 4 or more) of the desaturase or their gene products. A specificembodiment includes the cultivation in cell culture of a full or partialfatty acid auxotroph with supplementation of the fatty acid or a mixtureof fatty acids so as to increase the cell number, then allowing thecells to accumulate oil (e.g. to at least 40% by cell weight).Alternatively, the cells comprise a regulatable fatty acid synthesisgene that can be switched in activity. For example, the regulation canbe based on environmental conditions and the environmental conditionsduring a first, cell division, phase favor production of the fatty acidand the environmental conditions during a second, oil accumulation,phase disfavor production of the oil. For example, culture media pHand/or nitrogen levels can be used as an environmental control to switchexpression of a lipid pathway gene to produce a state of high or lowsynthetic enzyme activity. Examples of such cells are described inExample 7.

In a specific embodiment, a cell is cultivated using a modulation oflinoleic acid levels within the cell. In particular, the natural oil isproduced by cultivating the cells under a first condition that ispermissive to an increase in cell number due to the presence of linoleicacid and then cultivating the cells under a second condition that ischaracterized by linoleic acid starvation and thus is inhibitory to celldivision, yet permissive of oil accumulation. For example, a seedculture of the cells may be produced in the presence of linoleic acidadded to the culture medium. For example, the addition of linoleic acidto 0.25 g/L in the seed culture of a Prototheca strain deficient inlinoleic acid production due to ablation of two alleles of a fatty acylΔ12 desaturase (i.e., a linoleic auxotroph) was sufficient to supportcell division to a level comparable to that of wild type cells.Optionally, the linoleic acid can then be consumed by the cells, orotherwise removed or diluted. The cells are then switched into an oilproducing phase (e.g., supplying sugar under nitrogen limitingconditions such as described in WO2010/063032). Surprisingly, oilproduction has been found to occur even in the absence of linoleic acidproduction or supplementation, as demonstrated in the obligateheterotroph oleaginous microalgae Prototheca but generally applicable toother oleaginous microalgae, microorganisms, or even multicellularorganisms (e.g., cultured plant cells). Under these conditions, the oilcontent of the cell can increase to about 10, 20, 30, 40, 50, 60, 70,80, 90%, or more by dry cell weight, while the oil produced can havepolyunsaturated fatty acid (e.g.; linoleic+linolenic) profile with 5%,4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05% or less, as a percent oftotal triacylglycerol fatty acids in the oil. For example, the oilcontent of the cell can be 50% or more by dry cell weight and thetriglyceride of the oil produced less than 3% polyunsaturated fattyacids.

These oils can also be produced without the need (or reduced need) tosupplement the culture with linoleic acid by using cell machinery toproduce the linoleic acid, during the cell division phase, but less orno linoleic acid in the lipid production phase. The linoleic-producingcell machinery may be regulatable so as to produce substantially lesslinoleic acid during the oil producing phase. The regulation may be viamodulation of transcription of the desaturase gene(s) or modulation ofproduction of an inhibitor substance (e.g., regulated production ofhairpin RNA/RNAi). For example, the majority, and preferably all, of thefatty acid Δ12 desaturase activity can be placed under a regulatablepromoter regulated to express the desaturase in the cell division phase,but to be reduced or turned off during the oil accumulation phase. Theregulation can be linked to a cell culture condition such as pH, and/ornitrogen level, as described in the examples herein, or otherenvironmental condition. In practice, the condition may be manipulatedby adding or removing a substance (e.g., protons via addition of acid orbase) or by allowing the cells to consume a substance (e.g,nitrogen-supplying nutrients) to effect the desired switch in regulationof the desaturase activity.

Other genetic or non-genetic methods for regulating the desaturaseactivity can also be used. For example, an inhibitor of the desaturasecan be added to the culture medium in a manner that is effective toinhibit polyunsaturated fatty acids from being produced during the oilproduction phase.

Accordingly, in a specific embodiment of the invention, there is amethod comprising providing a recombinant cell having a regulatabledelta 12 fatty acid desaturase gene, under control of a recombinantregulatory element via an environmental condition. The cell iscultivated under conditions that favor cell multiplication. Uponreaching a given cell density, the cell media is altered to switch thecells to lipid production mode by nutrient limitation (e.g. reduction ofavailable nitrogen). During the lipid production phase, theenvironmental condition is such that the activity of the delta 12 fattyacid desaturase is downregulated. The cells are then harvested and,optionally, the oil extracted. Due to the low level of delta 12 fattyacid desaturase during the lipid production phase, the oil has lesspolyunsaturated fatty acids and has improved oxidative stability.Optionally the cells are cultivated heterotrophically and optionallymicroalgal cells.

Using one or more of these desaturase regulation methods, it is possibleto obtain a natural oil that it is believed has been previouslyunobtainable, especially in large scale cultivation in a bioreactor(e.g., more than 1000 L). The oil can have polyunsaturated fatty acidlevels that are 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or less, as anarea percent of total triacylglycerol fatty acids in the oil.

One consequence of having such low levels of polyunsaturates is thatoils are exceptionally stable to oxidation. Indeed, in some cases theoils may be more stable than any previously known natural cell oil. Inspecific embodiments, the oil is stable, without added antioxidants, at110° C. so that the inflection point in conductance is not yet reachedby 10 hours, 15 hours, 20 hours, 30 hours, 40, hours, 50 hours, 60hours, or 70 hours under conditions of the AOCS Cd 12b-92. Rancimattest, noting that for very stable oils, replenishment of water may berequired in such a test due to evaporation that occurs with such longtesting periods (see Example 5). For example the oil can have and OSIvalue of 40-50 hours or 41-46 hours at 110° C. without addedantioxidants. When antioxidants (suitable for foods or otherwise) areadded, the OSI value measured may be further increased. For example,with added tocopherol (100 ppm) and ascorbyl palmitate (500 ppm) or PANAand ascorbyl palmitate, such an oil can have an oxidative stabilityindex (OSI value) at 110° C. in excess 100 or 200 hours, as measured bythe Rancimat test. In another example, 1050 ppm of mixed tocopherols and500 pm of ascorbyl palmitate are added to an oil comprising less than 1%linoleic acid or less than 1% linoleic+linolenic acids; as a result, theoil is stable at 110° C. for 1, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, 12,13, 14, 15, or 16, 20, 30, 40 or 50 days, 5 to 15 days, 6 to 14 days, 7to 13 days, 8 to 12 days, 9 to 11 days, about 10 days, or about 20 days.The oil can also be stable at 130° C. for 1, 2, 3, 4, 5, 6, 7, 8, or 9,10, 11, 12, 13, 14, 15, or 16, 20, 30, 40 or 50 days, 5 to 15 days, 6 to14 days, 7 to 13 days, 8 to 12 days, 9 to 11 days, about 10 days, orabout 20 days. In a specific example, such an oil was found to be stablefor greater than 100 hours (about 128 hours as observed). In a furtherembodiment, the OSI value of the natural oil without added antioxidantsat 120° C. is greater than 15 hours or 20 hours or is in the range of10-15, 15-20, 20-25, or 25-50 hours, or 50-100 hours.

In an example, using these methods, the oil content of a microalgal cellis between 40 and about 85% by dry cell weight and the polyunsaturatedfatty acids in the fatty acid profile of the oil is between 0.001% and3% in the fatty acid profile of the oil and optionally yields a naturaloil having an OSI induction time of at least 20 hours at 110° C. withoutthe addition of antioxidants. In yet another example, there is a naturaloil produced by RBD treatment of a natural oil from an oleaginous cell,the oil comprises between 0.001% and 2% polyunsaturated fatty acids andhas an OSI induction time exceeding 30 hours at 110 C without theaddition of antioxidants. In yet another example, there is a natural oilproduced by RBD treatment of a natural oil from an oleaginous cell, theoil comprises between 0.001% and 1% polyunsaturated fatty acids and hasan OSI induction time exceeding 30 hours at 110 C without the additionof antioxidants.

In another specific embodiment there is an oil with reducedpolyunsaturate levels produced by the above-described methods. The oilis combined with antioxidants such as PANA and ascorbyl palmitate. Forexample, it was found that when such an oil was combined with 0.5% PANAand 500 ppm of ascorbyl palmitate the oil had an OSI value of about 5days at 130° C. or 21 days at 110° C. These remarkable results suggestthat not only is the oil exceptionally stable, but these twoantioxidants are exceptionally potent stabilizers of triglyceride oilsand the combination of these antioxidants may have general applicabilityincluding in producing stable biodegradable lubricants (e.g., jet enginelubricants). In specific embodiments, the genetic manipulation of fattyacyl Δ12 desaturase results in a 2 to 30, or 5 to 25, or 10 to 20 foldincrease in OSI (e.g., at 110° C.) relative to a strain without themanipulation. The oil can be produced by suppressing desaturase activityin a cell, including as described above.

Antioxidants suitable for use with the oils of the present inventioninclude alpha, delta, and gamma tocopherol (vitamin E), tocotrienol,ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid,β-carotene, lycopene, lutein, retinol (vitamin A), ubiquinol (coenzymeQ), melatonin, resveratrol, flavonoids, rosemary extract, propyl gallate(PG), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA),and butylated hydroxytoluene (BHT),N,N′-di-2-butyl-1,4-phenylenediamine,2,6-di-tert-butyl-4-methylphenol,2,4-dimethyl-6-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol,2,4-dimethyl-6-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol,2,6-di-tert-butylphenol, and phenyl-alpha-naphthylamine (PANA).

In addition to the desaturase modifications, in a related embodimentother genetic modifications may be made to further tailor the propertiesof the oil, as described throughout, including introduction orsubstitution of acyl-ACP thioesterases having altered chain lengthspecificity and/or overexpression of an endogenous or exogenous geneencoding a KAS, SAD, LPAAT, or DGAT gene. For example, a strain thatproduces elevated oleic levels may also produce low levels ofpolyunsaturates. Such genetic modifications can include increasing theactivity of stearoyl-ACP desaturase (SAD) by introducing an exogenousSAD gene, increasing elongase activity by introducing an exogenous KASIIgene, and/or knocking down or knocking out a FATA gene.

In a specific embodiment, a high oleic natural oil with lowpolyunsaturates may be produced. For example, the oil may have a fattyacid profile with greater than 60, 70, 80, 90, or 95% oleic acid andless than 5, 4, 3, 2, or 1% polyunsaturates. In related embodiments, anatural oil is produced by a cell having recombinant nucleic acidsoperable to decrease fatty acid Δ12 desaturase activity and optionallyfatty acid Δ15 desaturase so as to produce an oil having less than orequal to 3% polyunsaturated fatty acids with greater than 60% oleicacid, less than 2% polyunsaturated fatty acids and greater than 70%oleic acid, less than 1% polyunsaturated fatty acids and greater than80% oleic acid, or less than 0.5% polyunsaturated fatty acids andgreater than 90% oleic acid. It has been found that one way to increaseoleic acid is to use recombinant nucleic acids operable to decreaseexpression of a FATA acyl-ACP thioesterase and optionally overexpress aKAS II gene; such a cell can produce an oil with greater than or equalto 75% oleic acid. Alternately, overexpression of KASII can be usedwithout the FATA knockout or knockdown. Oleic acid levels can be furtherincreased by reduction of delta 12 fatty acid desaturase activity usingthe methods above, thereby decreasing the amount of oleic acid the isconverted into the unsaturates linoleic acid and linolenic acid. Thus,the oil produced can have a fatty acid profile with at least 75% oleicand at most 3%, 2%, 1%, or 0.5% linoleic acid. In a related example, theoil has between 80 to 95% oleic acid and about 0.001 to 2% linoleicacid, 0.01 to 2% linoleic acid, or 0.1 to 2% linoleic acid. Such oilswill have a low freezing point, with excellent stability and are usefulin foods, for frying, fuels, or in chemical applications. Further, theseoils may exhibit a reduced propensity to change color over time. In anillustrative chemical application, the high oleic oil is used to producea chemical. The oleic acid double bonds of the oleic acid groups of thetriglycerides in the oil can be epoxidized or hydroxylated to make apolyol. The epoxidized or hydroxylated oil can be used in a variety ofapplications. One such application is the production of polyurethane(including polyurethane foam) via condensation of the hydroxylatedtriglyceride with an isocyanate, as has been practiced with hydroxylatedsoybean oil or castor oil. See, e.g. US2005/0239915, US2009/0176904,US2005/0176839, US2009/0270520, and U.S. Pat. No. 4,264,743 andZlatanic, et al, Biomacromolecules 2002, 3, 1048-1056 (2002) forexamples of hydroxylation and polyurethane condensation chemistries.Suitable hydroxyl forming reactions include epoxidation of one or moredouble bonds of a fatty acid followed by acid catalyzed epoxide ringopening with water (to form a diol), alcohol (to form a hydroxyl ether),or an acid (to form a hydroxyl ester). There are multiple advantages ofusing the high-oleic/low polyunsaturated oil in producing a bio-basedpolyurethane: (1) the shelf-life, color or odor, of polyurethane foamsmay be improved; (2) the reproducibility of the product may be improveddue to lack of unwanted side reactions resulting from polyunsaturates;(3) a greater degree of hydroxylation reaction may occur due to lack ofpolyunsaturates and the structural characteristics of the polyurethaneproduct can be improved accordingly.

The low polyunsaturated or high oleic/low polyunsaturated oils describedhere may be advantageously used in chemical applications where yellowingis undesirable. For example, yellowing can be undesirable in paints orcoatings made from the triglycerides fatty acids derived from thetriglycerides. Yellowing may be caused by reactions involvingpolyunsaturated fatty acids and tocotrienols and/or tocopherols. Thus,producing the high stability oil in an oleaginous microbe with lowlevels of tocotrienols can be advantageous in elevating high colorstability a chemical composition made using the oil. In contrast tocommonly used plant oils, through appropriate choice of oleaginousmicrobe, the natural oils of these embodiments can have tocopherols andtocotrienols levels of 1 g/L or less. In a specific embodiment, anatural oil has a fatty acid profile with less than 2% withpolyunsaturated fatty acids and less than 1 g/L for tocopherols,tocotrienols or the sum of tocopherols and tocotrienols. In anotherspecific embodiment, the natural oil has a fatty acid profile with lessthan 1% with polyunsaturated fatty acids and less than 0.5 g/L fortocopherols, tocotrienols or the sum of tocopherols and tocotrienols

Any of the high-stability (low-polyunsaturate) natural oils orderivatives thereof can be used to formulate foods, drugs, vitamins,nutraceuticals, personal care or other products, and are especiallyuseful for oxidatively sensitive products. For example, thehigh-stability natural oil (e.g., less than or equal to 3%, 2% or 1%polyunsaturates) can be used to formulate a sunscreen (e.g. acomposition having one or more of avobenzone, homosalate, octisalate,octocrylene or oxybenzone) or retinoid face cream with an increasedshelf life due to the absence of free-radical reactions associated withpolyunsaturated fatty acids. For example, the shelf-life can beincreased in terms of color, odor, organoleptic properties or % activecompound remaining after accelerated degradation for 4 weeks at 54° C.The high stability oil can also be used as a lubricant with excellenthigh-temperature stability. In addition to stability, the oils can bebiodegradable, which is a rare combination of properties.

In another related embodiment, the fatty acid profile of a natural oilis elevated in C8 to C16 fatty acids through additional geneticmodification, e.g. through overexpression of a short-chain to mid chainpreferring acyl-ACP thioesterase or other modifications described here.A low polyunsaturated oil in accordance with these embodiments can beused for various industrial, food, or consumer products, including thoserequiring improved oxidative stability. In food applications, the oilsmay be used for frying with extended life at high temperature, orextended shelf life.

Where the oil is used for frying, the high stability of the oil mayallow for frying without the addition of antioxidant and/or defoamers(e.g. silicone). As a result of omitting defoamers, fried foods mayabsorb less oil. Where used in fuel applications, either as atriglyceride or processed into biodiesel or renewable diesel (see, e.g.,WO2008/151149 WO2010/063032, and WO2011/150410), the high stability canpromote storage for long periods, or allow use at elevated temperatures.For example, the fuel made from the high stability oil can be stored foruse in a backup generator for more than a year or more than 5 years. Thefrying oil can have a smoke point of greater than 200° C., and freefatty acids of less than 0.1% (either as a natural oil or afterrefining).

The low polyunsaturated oils may be blended with food oils, includingstructuring fats such as those that form beta or beta prime crystals,including those produced as described below. These oils can also beblended with liquid oils. If mixed with an oil having linoleic acid,such as corn oil, the linoleic acid level of the blend may approximatethat of high oleic plant oils such as high oleic sunflower oils (e.g.,about 80% oleic and 8% linoleic).

Blends of the low polyunsaturated natural oil can be interesterifiedwith other oils. For example, the oil can be chemically or enzymaticallyinteresterified. In a specific embodiment, a low polyunsaturated oilaccording to an embodiment of the invention has at least 10% oleic acidin its fatty acid profile and less than 5% polyunsaturates and isenzymatically interesterified with a high saturate oil (e.g.hydrogenated soybean oil or other oil with high stearate levels) usingan enzyme that is specific for sn-1 and sn-2 triacylglycerol positions.The result is an oil that includes a stearate-oleate-stearate (SOS).Methods for interesterification are known in the art; see for example,“Enzymes in Lipid Modification,” Uwe T. Bornschuer, ed., Wiley_VCH,2000, ISBN 3-527-30176-3.

High stability oils can be used as spray oils. For example, dried fruitssuch as raisins can be sprayed with a high stability oil having lessthan 5, 4, 3, 2, or 1% polyunsaturates. As a result, the spray nozzleused will become clogged less frequently due to polymerization oroxidation product buildup in the nozzle that might otherwise result fromthe presence of polyunsaturates.

In a further embodiment, an oil that is high is SOS, such as thosedescribed below can be improved in stability by knockdown or regulationof delta 12 fatty acid desaturase.

Oils that contain high amounts of polyunsaturated fatty acids, e.g., 7%or higher, are less stable than oils with lower amounts of PUFAs.Polyunsaturated fatty acids contain two or more double bonds. PUFAs aremore susceptible to oxidative degradation and/or polymerization thanPUFAs that contain only one double bond and are also more susceptible tooxidative degradation and/or polymerization than fully saturated fattyacids. Oils with lower amounts of PUFAs are more robust and consequentlyare more desirable in applications where stability is a concern. Suchapplications include those were the oil is exposed to high and/orprolonged heat, light, and oxidative conditions.

Spray oils comprising the high oleic, low PUFA oils of the invention areapplied to various products including human and animal foods. As an oilmoves through the orifice during the spraying process, oils that havePUFAs polymerize and form a film, thereby clogging the orifice. Oilsthat comprise more than 7% PUFAs polymerize more readily than oils thatcontain less than 7% PUFAs. The high oleic, low PUFA oils of the presentinvention reduce clogging of the orifices that the oil is sprayed from.The spraying device therefor can operate for longer periods betweenmaintenance cycles.

A release agent comprising the high oleic, low PUFA oils of theinvention are used during manufacturing of a product to facilitaterelease of the product from the mold. During the manufacturing process,oils that have PUFAs degrade and/or polymerize to form a film. Thedegradation products and film formed from the PUFAs inhibit the releaseof the product from the mold. The degradation products and film formedfrom the PUFAs also adversely impact the finished product by discoloringthe product or imparting an unacceptable odor. Oils that comprise morethan 7% PUFAs degrade and/or polymerize more readily than oils thatcontain less than 7% PUFAs. The high oleic, low PUFA oils of the presentinvention minimizes the discoloration and odor imparted to the productas compared to a release agent comprising more than 7% PUFAs.

A heat transfer fluid comprising the high oleic, low PUFA oils of theinvention provide longer service life than a heat transfer fluidcomprising conventional triglyceride oils that contain higher amounts ofPUFA (e.g., vegetable oil or a high oleic vegetable oil). The longerservice life results from the low amounts of PUFAs. As a heat transferfluid dissipates heat generated during the operation of the device forexample, a dielectric fluid in a high voltage transformer or a computerserver coolant, the PUFAs oxidize and form degradation products, and/orthe PUFAs polymerize forming films. Conventional triglyceride oils thatcomprise more than 7% PUFAs degrade and/or polymerize more readily thanoils that contain less than 7% PUFAs. The heat transfer fluidscomprising high oleic, low PUFA oils are more resistant to oxidation andpolymerization during the service life of the fluid. Heat transferfluids comprising high oleic, low PUFA oils of the present inventionprovide longer service life as compared to a heat transfer fluidscomprising triglyceride oils that contain more than 7% PUFAs.

A hydraulic fluid comprising the high oleic, low PUFA oils of theinvention provide longer service life than a hydraulic fluid comprisingconventional triglyceride oils. During use, lubricants that comprisingPUFAs degrade and/or polymerize to form a film. The degradation productsand film formed from the PUFAs decreases the lubricity of the lubricant.Conventional oils that comprise more than 7% PUFAs degrade and/orpolymerize more readily than oils that contain less than 7% PUFAs. Thehigh oleic, low PUFA oils of the present invention provides longerservice life as compared to a lubricant comprising more than 7% PUFAs.

A hydraulic fluid comprising the high oleic, low PUFA oils of theinvention provide longer service life than a hydraulic fluid comprisingconventional triglyceride oils. During use, lubricants that comprisingPUFAs degrade and/or polymerize to form a film. The degradation productsand film formed from the PUFAs decreases the lubricity of the lubricant.Conventional oils that comprise more than 7% PUFAs degrade and/orpolymerize more readily than oils that contain less than 7% PUFAs. Thehigh oleic, low PUFA oils of the present invention provides longerservice life as compared to a lubricant comprising more than 7% PUFAs.

A lubricant comprising the high oleic, low PUFA oils of the inventionprovide longer service life than a lubricant comprising conventionaltriglyceride oils that contain higher amounts of PUFA (e.g., vegetableoil or a high oleic vegetable oil). During use, lubricants thatcomprising PUFAs degrade and/or polymerize to form a film. Thedegradation products and film formed from the PUFAs decreases thelubricity of the lubricant. Conventional triglyceride oils that comprisemore than 7% PUFAs degrade and/or polymerize more readily than oils thatcontain less than 7% PUFAs. Lubricants comprising high oleic, low PUFAoils of the present invention provide longer service life as compared toa lubricant comprising triglyceride oils that contain more than 7%PUFAs.

A polyurethane is a compound that comprises a carbamate (urethane)linkage. In some embodiments the polyurethane is a polymer of organicunits. The polymer is prepared by the reaction of a first organic unitcomprising an isocyanate moiety (e.g., C(O)N—R₁—NC(O)) and a secondorganic unit comprising a hydroxyl group (e.g., HO—R₂—OH) or an epoxidegroup (e.g., (—CH(O)CH—R₂—CH(O)HC—). A polyurethane can be representedas —[C(O)NH—R₁—NHC(O)—O—R₂—O]m-, wherein the subscript m is a numberthat denotes the number of monomers contained in the polymer. R₁ and R₂can be the same or different, but are typically different. Polyurethanesare used in many different applications including both flexible andrigid materials. Polyurethanes are used in shoes, automobiles,airplanes, bushings, gaskets, adhesives, carpeting, spandex fibers,housing for electronics and the like.

In yet another embodiment provided is a composition prepared by reactinga hydroxylated oil, an epoxidated oil and/or a hydroxylated fatty acid,or epoxidated fatty acid with a compound that contains an isocyanatemoiety to form a polyurethane

In one embodiment provided is a composition prepared by reacting apolyol (e.g., hydroxylated oil and/or a hydroxylated fatty acid) or anepoxidated oil or expoxidated fatty acid with a compound that containsan isocyanate moiety. The unsaturated portions of the fatty acid or oilare hydroxylated or epoxidated first by well-known chemistries prior toreaction with an isocyanate. Polyurethanes using castor oil and anisocyanate have been produced. Polyurethanes are ubiquitous in theproducts we use today. Polyurethanes are found in automobiles, toys,athletic equipment, consumer electronics, shoes, mattresses, cushions,adhesives, construction materials, and the like. Currently,polyurethanes made with castor oil are commercially available from BASF,Itoh Oil and others. Polyurethanes made with hydroxylated soybean oilare commercially available from Cargill, Dow, Bayer and others.

In various embodiments, the compositions provided herein include one ormore additive(s), such as an antioxidant, a metal ion deactivator, acorrosion inhibitor, moisture scavenger, a demulsifier, an anti-wearadditive, a pour point depressant, or an anti-hydrolysis compound.

In various embodiments the compositions provided herein further compriseone or more of (a) an antioxidant, including but not limited totocopherols, BHT and other phenols; (b) a deactivator of metal ions suchas Cu, Zn, and the like, including but not limited to a benzotriazole;(c) corrosion inhibitors, including but not limited to ester sulfonatesand succinic acid esters; (d) demulsifiers; (e) anti-wear additives,including but not limited to zinc dithiophosphate; (f) additives todepress the pour point, including but not limited to malan styrenecopolymers and poly(alkyl)acrylates, including but not limited topolymethacrylates; and (g) compounds that protect against hydrolysis,including but not limited to carbodiimides, (h) a pour point depressant,including but not limited to VISCOPLEX® 10-310 or 1-133 (Rohmax-EvonikAdditives GmbH), or other poly(alkyl) acrylates andpoly(methyl)acrylates such as INFINEUM® V-351 (Infineum UK limited),PMA-D110 and PMA D; (i) a carbodiimide; (j) a synthetic ester; (k) polyalpha olefins (PAO); or (k) ester of estolides.

Optionally, other additives for increasing the oxidative stability ofthe isolated lipids can be admixed with the microbial oil, lubricant, ordielectric fluid produced by these methods. Examples of such additivesinclude antioxidants such as tocopherols (vitamin E, e.g., alpha-, beta-and/or delta-tocopherol), ascorbic acid (vitamin C). Suitableanti-oxidants are commercially available. The BASF company markets aline of suitable phenol based and amine based antioxidants under thebrand name IRGANOX®. IRGANOX L109, IRGANOX L64, IRGANOX L57, otherIRGANOX antioxidants, and other phenol based and amine based compoundsare suitable as antioxidant additives to the oils and products includingdielectric fluids. Other nonlimiting examples of antioxidants includebutylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT),mono-tertiary butyl hydro quinone (TBHQ), butylated hydroanisole,tetrahydrobutrophenone, ascorbyl palmitate, and propyl gallate. Incertain embodiments, a microbial oil-based product, e.g., a dielectricfluid, additionally includes an antioxidant at 0.1% to 5% by weight, andpreferably at 0.5% to 2%.

Useful oxygen scavenging compounds include those commonly employed inthe food packaging industry. Representative of the oxygen scavengingcompounds useful in the practice of the invention include the following:sodium sulfite; copper sulfate pentahydrate; a combination of carbon andactivated iron powder; mixtures of hydrosulfite, calcium hydroxide,sodium bicarbonate and activated carbon; a metal halide powder coated onthe surface of a metal powder; and combinations of alkali compounds,such as calcium hydroxide, with sodium carbonate or sodium bicarbonate.Mixtures and combinations of one or more of the above compositions arealso considered useful. Also useful as oxygen scavenging compounds arethose compositions provided according to U.S. Pat. No. 2,825,651, whichis incorporated by reference, including an oxygen remover compositioncomprising an intermixing of a sulfite salt and an accelerator such ashydrated copper sulfate, stannous chloride, or cobaltous oxide. Anotheruseful class of oxygen scavenging compounds includes those compositionscomprising a salt of manganese, iron, cobalt or nickel, an alkalicompound, and a sulfite or deliquescent compound, such as disclosed byU.S. Pat. No. 4,384,972, which also is incorporated by reference.Preferred oxygen scavenging compounds include (or include as their basecomponent) at least one basic iron oxide, such as a ferrous iron oxide,or are made of mixtures of iron oxide materials. Useful ironoxide-containing compositions are available commercially, for example,under the “Ageless” trade name from the Mitsubishi Gas Chemical Companyof Duncan, S.C. and under the “Freshmax” trade name from MultisorbTechnologies, Inc. of Buffalo, N.Y. Also useful are oxygen absorbingagents comprising a mixture of ferrous salts and an oxidation modifierand/or a metallic sulfite or sulfate compound.

Other additives that can be optionally added to the lipids for use asproducts such as spray oil, dielectric fluid, heat transfer fluid,release agent, lubricant, hydraulic fluid, surfactant, solvent,architectural coating, or a personal care product are deactivators formetal ions, corrosion inhibitors, anti-wear additives, and/or hydrolysisprotectants. Some widely used additives are described in Schneider,2006, J Science Food and Agriculture; 86: 1769-1780). Metal iondeactivators (e.g., metal chelators) have two main functions. Theysuppress chemical attack on the surface of the metal and they alsopassivate the metal surface to suppress any residues that may act ascatalysts for radical (unpaired electron) formation. Metal deactivatorsare commercially available. For example, the BASF company provides aline of metal deactivators, including the IRGAMET® line of metaldeactivators. The RTVANDERBILT company sells the CUVAN® line of metaldeactivators. Other examples of metal deactivators include derivatizedtriazoles including 1-(di-isooctylaminomethyl)-1,2,4-triazole,1-(2-methoxyprop-2-yl)tolyltriazole,1-(1-cyclohexyloxypropyl)tolyltriazole,1-(1-cyclohexyloxyheptyl)tolyltriazole,1-(1-cyclohexyloxybutyl)tolyltriazole,1-[bis(2-ethylhexyl)aminomethyl-4-methylbenzotriazole, derivatizedborons including triethyl borate, tripropyl borate, triisopropyl borate,tributyl borate, tripentyl borate, trihexyl borate, tricyclohexylborate, trioctyl borate, triisooctyl borate, and other derivatizedhydrazine metal deactivator, e.g.,2′,3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]proponiohydrazine,and the like. In certain embodiments, a microbial oil-based product ofthe present invention including spray oil, dielectric fluid, heattransfer fluid, release agent, lubricant, hydraulic fluid, surfactant,solvent, architectural coating, or a personal care product additionallyincludes one or more metal deactivators at 0.1% to 5% by weight, andpreferably at 0.5% to 2%.

V. Cells with Exogenous Acyltransferases

In various embodiments of the present invention, one or more genesencoding an acyltransferase (an enzyme responsible for the condensationof a fatty acid with glycerol or a glycerol derivative to form anacylglyceride) can be introduced into an oleaginous cell (e.g., aplastidic microalgal cell) so as to alter the fatty acid composition ofa natural oil produced by the cell. The genes may encode one or more ofa glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acidacyltransferase (LPAAT), also known as 1-acylglycerol-3-phosphateacyltransferase (AGPAT), phosphatidic acid phosphatase (PAP), ordiacylglycerol acyltransferase (DGAT) that transfers an acyl group tothe sn-3 position of DAG, thereby producing a TAG.

Recombinant nucleic acids may be integrated into a plasmid or chromosomeof the cell. Alternately, the gene encodes an enzyme of a lipid pathwaythat generates TAG precursor molecules through fattyacyl-CoA-independent routes separate from that above. Acyl-ACPs may besubstrates for plastidial GPAT and LPAAT enzymes and/or mitochondrialGPAT and LPAAT enzymes. Among further enzymes capable of incorporatingacyl groups (e.g., from membrane phospholipids) to produce TAGs isphospholipid diacylglycerol acyltransferase (PDAT). Still furtheracyltransferases, including lysophosphosphatidylcholine acyltransferase(LPCAT), lysophosphosphatidylserine acyltransferase (LPSAT),lysophosphosphatidylethanolamine acyltransferase (LPEAT), andlysophosphosphatidylinositol acyltransferase (LPIAT), are involved inphospholipid synthesis and remodeling that may impact triglyceridecomposition.

The exogenous gene can encode an acyltransferase enzyme havingpreferential specificity for transferring an acyl substrate comprising aspecific number of carbon atoms and/or a specific degree of saturationis introduced into a oleaginous cell so as to produce an oil enriched ina given regiospecific triglyceride. For example, the coconut (Cocosnucifera) lysophosphatidic acid acyltransferase has been demonstrated toprefer C12:0-CoA substrates over other acyl-CoA substrates (Knutzon etal., Plant Physiology, Vol. 120, 1999, pp 739-746), whereas the1-acyl-sn-3-glycerol-3-phosphate acyltransferase of maturing safflowerseeds shows preference for linoleoyl-CoA and oleoyl-CoA substrates overother acyl-CoA substrates, including stearoyl-CoA (Ichihara et al.,European Journal of Biochemistry, Vol. 167, 1989, pp 339-347).Furthermore, acyltransferase proteins may demonstrate preferentialspecificity for one or more short-chain, medium-chain, or long-chainacyl-CoA or acyl-ACP substrates, but the preference may only beencountered where a particular, e.g. medium-chain, acyl group is presentin the sn-1 or sn-3 position of the lysophosphatidic acid donorsubstrate. As a result of the exogenous gene, a TAG oil can be producedby the cell in which a particular fatty acid is found at the sn-2position in greater than 20, 30, 40, 50, 60, 70, 90, or 90% of the TAGmolecules.

In some embodiments of the invention, the cell makes an oil rich insaturated-unsaturated-saturated (sat-unsat-sat) TAGs. Sat-unsat-sat TAGSinclude 1,3-dihexadecanoyl-2-(9Z-octadecenoyl)-glycerol (referred to as1-palmitoyl-2-oleyl-glycero-3-palmitoyl),1,3-dioctadecanoyl-2-(9Z-octadecenoyl)-glycerol (referred to as1-stearoyl-2-oleyl-glycero-3-stearoyl), and1-hexadecanoyl-2-(9Z-octadecenoyl)-3-octadecanoy-glycerol (referred toas 1-palmitoyl-2-oleyl-glycero-3-stearoyl). These molecules are morecommonly referred to as POP, SOS, and POS, respectively, where ‘P’represents palmitic acid, ‘S’ represents stearic acid, and ‘O’represents oleic acid. Further examples ofsaturated-unsaturated-saturated TAGs include MOM, LOL, MOL, COC and COL,where ‘M’ represents myristic acid, 1′ represents lauric acid, and ‘C’represents capric acid (C8:0). Trisaturates, triglycerides with threesaturated fatty acyl groups, are commonly sought for use in foodapplications for their greater rate of crystallization than other typesof triglycerides. Examples of trisaturates include PPM, PPP, LLL, SSS,CCC, PPS, PPL, PPM, LLP, and LLS. In addition, the regiospecificdistribution of fatty acids in a TAG is an important determinant of themetabolic fate of dietary fat during digestion and absorption.

According to certain embodiments of the present invention, oleaginouscells are transformed with recombinant nucleic acids so as to producenatural oils that comprise an elevated amount of a specifiedregiospecific triglyceride, for example 1-acyl-2-oleyl-glycero-3-acyl,or 1-acyl-2-lauric-glycero-3-acyl where oleic or lauric acidrespectively is at the sn-2 position, as a result of introducedrecombinant nucleic acids. Alternately, caprylic, capric, myristic, orpalmitic acid may be at the sn-2 position. The amount of the specifiedregiospecific triglyceride present in the natural oil may be increasedby greater than 5%, greater than 10%, greater than 15%, greater than20%, greater than 25%, greater than 30%, greater than 35%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, greater than 100-500%, or greater than 500% thanin the natural oil produced by the microorganism without the recombinantnucleic acids. As a result, the sn-2 profile of the cell triglyceridemay have greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90% of theparticular fatty acid.

The identity of the acyl chains located at the distinct stereospecificor regiospecific positions in a glycerolipid can be evaluated throughone or more analytical methods known in the art (see Luddy et al., J.Am. Oil Chem. Soc., 41, 693-696 (1964), Brockerhoff, J. Lipid Res., 6,10-15 (1965), Angers and Aryl, J. Am. Oil Chem. Soc., Vol. 76:4, (1999),Buchgraber et al., Eur. J. Lipid Sci. Technol., 106, 621-648 (2004)), orin accordance with Examples 1, 2, and 8 given below.

The positional distribution of fatty acids in a triglyceride moleculecan be influenced by the substrate specificity of acyltransferases andby the concentration and type of available acyl moieties substrate pool.Nonlimiting examples of enzymes suitable for altering theregiospecificity of a triglyceride produced in a recombinantmicroorganism are listed in Tables 1-4. One of skill in the art mayidentify additional suitable proteins.

TABLE 1 Glycerol-3-phosphate acyltransferases and GenBank accessionnumbers. glycerol-3-phosphate acyltransferase Arabidopsis BAA00575thaliana glycerol-3-phosphate acyltransferase Chlamydomonas EDP02129reinhardtii glycerol-3-phosphate acyltransferase Chlamydomonas Q886Q7reinhardtii acyl-(acyl-carrier-protein): Cucurbita moschata BAB39688glycerol-3-phosphate acyltransferase glycerol-3-phosphateacyltransferase Elaeis AAF64066 guineensis glycerol-3-phosphateacyltransferase Garcina ABS86942 mangostana glycerol-3-phosphateacyltransferase Gossypium hirsutum ADK23938 glycerol-3-phosphateacyltransferase Jatropha curcas ADV77219 plastid glycerol-3-phosphateJatropha curcas ACR61638 acyltransferase plastidial glycerol-phosphateRicinus communis EEF43526 acyltransferase glycerol-3-phosphateacyltransferase Vica faba AAD05164 glycerol-3-phosphate acyltransferaseZea mays ACG45812

Lysophosphatidic acid acyltransferases suitable for use with themicrobes and methods of the invention include, without limitation, thoselisted in Table 2.

TABLE 2 Lysophosphatidic acid acyltransferases and GenBank accessionnumbers. 1-acyl-sn-glycerol-3-phosphate Arabidopsis thaliana AEE85783acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica juncea ABQ42862acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica juncea ABM92334acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica napus CAB09138acyltransferase lysophosphatidic acid Chlamydomonas EDP02300acyltransferase reinhardtii lysophosphatidic acid Limnanthes albaAAC49185 acyltransferase 1-acyl-sn-glycerol-3-phosphate Limnanthesdouglasii CAA88620 acyltransferase (putative)acyl-CoA:sn-1-acylglycerol-3- Limnanthes douglasii ABD62751 phosphateacyltransferase 1-acylglycerol-3-phosphate O- Limnanthes douglasiiCAA58239 acyltransferase 1-acyl-sn-glycerol-3-phosphate Ricinus communisEEF39377 acyltransferase

Diacylglycerol acyltransferases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 3.

TABLE 3 Diacylglycerol acyltransferases and GenBank accession numbers.diacylglycerol acyltransferase Arabidopsis CAB45373 thalianadiacylglycerol acyltransferase Brassica juncea AAY40784 putativediacylglycerol acyltransferase Elaeis guineensis AEQ94187 putativediacylglycerol acyltransferase Elaeis guineensis AEQ94186 acylCoA:diacylglycerol acyltransferase Glycine max AAT73629 diacylglycerolacyltransferase Helianthus annus ABX61081 acyl-CoA:diacylglycerol Oleaeuropaea AAS01606 acyltransferase 1 diacylglycerol acyltransferaseRicinus AAR11479 communis

Phospholipid diacylglycerol acyltransferases suitable for use with themicrobes and methods of the invention include, without limitation, thoselisted in Table 4.

TABLE 4 Phospholipid diacylglycerol acyltransferases and GenBankaccession numbers. phospholipid:diacylglycerol Arabidopsis AED91921acyltransferase thaliana Putative phospholipid:diacylglycerol ElaeisAEQ94116 acyltransferase guineensis phospholipid:diacylglycerol Glycinemax XP_003541296 acyltransferase 1-like phospholipid:diacylglycerolJatropha AEZ56255 acyltransferase curcas phospholipid:diacylglycerolRicinus ADK92410 acyltransferase communis phospholipid:diacylglycerolRicinus AEW99982 acyltransferase communis

In an embodiment of the invention, known or novel LPAAT genes aretransformed into the oleaginous cells so as to alter the fatty acidprofile of triglycerides produced by those cells, most notably byaltering the sn-2 profile of the triglycerides. For example, by virtueof expressing an exogenous active LPAAT in an oleaginous cell, thepercent of unsaturated fatty acid at the sn-2 position is increased by10, 20, 30, 40, 50, 60, 70, 80, 90% or more. For example, a cell mayproduce triglycerides with 30% unsaturates (which may be primarily 18:1and 18:2 and 18:3 fatty acids) at the sn-2 position. In this example,introduction of the LPAAT activity increases the unsaturates at the sn-2position by 20% so that 36% of the triglycerides comprise unsaturates atthe sn-2 position. Alternately, an exogenous LPAAT can be used toincrease mid-chain fatty acids including saturated mid-chains such asC8:0, C10:0, C12:0, C14:0 or C16:0 moieties at the sn-2 position. As aresult, mid-chain levels in the overall fatty acid profile may beincreased. Examples 43 and 44 describe altering the sn-2 and fatty acidprofiles in an oleaginous microbe. As can be seen from those examples,the choice of LPAAT gene is important in that different LPAATs can causea shift in the sn-2 and fatty acid profiles toward different acyl groupchain-lengths or saturation levels. For example, the LPAAT of Example 43increases C10-C14 fatty acids and the LPAAT of Example 44 causes anincrease in C16 and C18 fatty acids. As in these examples, introductionof an exogenous LPAAT can be combined with introduction of exogenousacyl-ACP thioesterase. Combining a mid-chain preferring LPAAT and amid-chain preferring FatB was found to give an additive effect; thefatty acid profile was shifted more toward the mid-chain fatty acidswhen both an exogenous LPAAT and FatB gene was present than when only anexogenous FatB gene was present. In a specific embodiment, the oilproduced by a cell comprising an exogenous mid-chain specific LPAAT and(optionally) an exogenous FatB acyl-ACP thioesterase gene can have afatty acid profile with 40, 50, 60, 70, 80% or more of C8:0, C10:0,C12:0, C14:0, or C16:0 fatty acids (separately or in sum).

Specific embodiments of the invention are a nucleic acid construct, acell comprising the nucleic acid construct, a method of cultivating thecell to produce a triglyceride, and the triglyceride oil produced wherethe nucleic acid construct has a promoter operably linked to a novelLPAAT coding sequence. The coding sequence can have an initiation codonupstream and a termination codon downstream followed by a 3′ UTRsequence. In a specific embodiment, the LPAAT gene has LPAAT activityand a coding sequence have at least 75, 80, 85, 90, 95, 96, 97, 98 or98% sequence identity to any of the cDNAs of SEQ ID NOs: 80 to 85 or afunctional fragment thereof including equivalent sequences by virtue ofdegeneracy of the genetic code. Introns can be inserted into thesequence as well. Alternately, the LPAAT gene codes for the amino acidsequence of SEQ ID NOs 77-79 or functional fragments thereof, or aprotein having at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% aminoacid sequence identity. Plants expressing the novel LPAAT are expresslyincluded in the embodiments and can be produced using known geneticengineering techniques.

VI. Cells with Exogenous Elongases or Elongase Complex Enzymes

In various embodiments of the present invention, one or more genesencoding elongases or components of the fatty acyl-CoA elongationcomplex can be introduced into an oleaginous cell (e.g., a plastidicmicroalgal cell) so as to alter the fatty acid composition of the cellor of a natural oil produced by the cell. The genes may encode abeta-ketoacyl-CoA synthase (also referred to as 3-ketoacyl synthase,beta-ketoacyl synthase or KCS), a ketoacyl-CoA reductase, ahydroxyacyl-CoA dehydratase, enoyl-CoA reductase, or elongase. Theenzymes encoded by these genes are active in the elongation of acyl-coAmolecules liberated by acyl-ACP thioesterases. Recombinant nucleic acidsmay be integrated into a plasmid or chromosome of the cell. In aspecific embodiment, the cell is of Chlorophyta, including heterotrophiccells such as those of the genus Prototheca.

Beta-Ketoacyl-CoA synthase and elongase enzymes suitable for use withthe microbes and methods of the invention include, without limitation,those listed in Table 5.

TABLE 5 Beta-Ketoacyl-CoA synthases and elongases listed with GenBankaccession numbers. Trypanosoma brucei elongase 3 (GenBank Accession No.AAX70673), Marchanita polymorpha (GenBank Accession No. AAP74370),Trypanosoma cruzi fatty acid elongase, putative (GenBank Accession No.EFZ33366), Nannochloropsis oculata fatty acid elongase (GenBankAccession No. ACV21066.1), Leishmania donovani fatty acid elongase,putative (GenBank Accession No. CBZ32733.1), Glycine max 3-ketoacyl-CoAsynthase 11-like (GenBank Accession No. XP_003524525.1), Medicagotruncatula beta-ketoacyl-CoA synthase (GenBank Accession No.XP_003609222), Zea mays fatty acid elongase (GenBank Accession No.ACG36525), Gossypium hirsutum beta-ketoacyl-CoA synthase (GenBankAccession No. ABV60087), Helianthus annuus beta-ketoacyl-CoA synthase(GenBank Accession No. ACC60973.1), Saccharomyces cerevisiae ELO1(GenBank Accession No. P39540), Simmondsia chinensis beta-ketoacyl-CoAsynthase (GenBank Accession No. AAC49186), Tropaeolum majus putativefatty acid elongase (GenBank Accession No. AAL99199, Brassica napusfatty acid elongase (GenBank Accession No. AAA96054)

In an embodiment of the invention, an exogenous gene encoding abeta-ketoacyl-CoA synthase or elongase enzyme having preferentialspecificity for elongating an acyl substrate comprising a specificnumber of carbon atoms and/or a specific degree of acyl chain saturationis introduced into a oleaginous cell so as to produce a cell or an oilenriched in fatty acids of specified chain length and/or saturation.Example 40 describes engineering of Prototheca strains in whichexogenous fatty acid elongases with preferences for extending midchainfatty acyl-CoAs have been overexpressed to increase the concentration ofstearate. Examples 42 and 54 describe engineering of Prototheca in whichexogenous elongases or beta-ketoacyl-CoA synthases with preferences forextending monounsaturated and saturated C18- and C20-CoA substrates areoverexpressed to increase the concentration of erucic acid.

In specific embodiments, the oleaginous cell produces an oil comprisinggreater than 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60 70, or 80% erucicand/or eicosenoic acid. Alternately, the cell produces an oil comprising0.5-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, or 90-99% erucic or eicosenoic acid. The cell may compriserecombinant acids described above in connection with high-oleic oilswith a further introduction of an exogenous beta-ketoacyl-CoA synthasethat is active in elongating oleoyl-CoA. As a result of the expressionof the exogenous beta-ketoacyl-CoA synthase, the natural production oferucic or eicosenoic acid by the cell can be increased by more than 2,3, 4, 5, 10, 20, 30, 40, 50, 70, 100, 130, 170 or 200 fold. The higherucic and/or eicosenoic oil can also be a high stability oil; e.g., onecomprising less than 5, 4, 3, 2, or 1% polyunsaturates and/or having theOSI values described in Section IV of this application and accompanyingExamples. In a specific embodiment, the cell is a microalgal cell,optionally cultivated heterotrophically. As in the other embodiments,the oil/fat can be produced by genetic engineering of a plastidic cell,including heterotrophic microalgae of the phylum Chlorophyta, the classTrebouxiophytae, the order Chlorellales, or the family Chlorellacae.Preferably, the cell is oleaginous and capable of accumulating at least40% oil by dry cell weight. The cell can be an obligate heterotroph,such as a species of Prototheca, including Prototheca moriformis orPrototheca zopfii.

VII. Regiospecific and Stereospecific Oils/Fats

In an embodiment, a recombinant cell produces a natural fat or oilhaving a given regiospecific makeup. As a result, the cell can producetriglyceride fats having a tendency to form crystals of a givenpolymorphic form; e.g., when heated to above melting temperature andthen cooled to below melting temperature of the fat. For example, thefat may tend to form crystal polymorphs of the β or β′ form (e.g., asdetermined by X-ray diffraction analysis), either with or withouttempering. The fats may be ordered fats. In specific embodiments, thefat may directly from either β or β′ crystals upon cooling;alternatively, the fat can proceed through a β form to a β′ form. Suchfats can be used as structuring laminating or coating fats for foodapplications. The natural fats can be incorporated into candy, dark orwhite chocolate, chocolate flavored confections, ice cream, margarinesor other spreads, cream fillings, pastries, or other food products.Optionally, the fats can be semi-solid (at room temperature) yet free ofartificially produced trans-fatty acids. Such fats can also be useful inskin care and other consumer or industrial products. PCT/US2013/037261describes regiospecific triglycerides and their production.

As in the other embodiments, the fat can be produced by geneticengineering of a plastidic cell, including heterotrophic eukaryoticmicroalgae of the phylum Chlorophyta, the class Trebouxiophytae, theorder Chlorellales, or the family Chlorellacae. Preferably, the cell isoleaginous and capable of accumulating at least 40% oil by dry cellweight. The cell can be an obligate heterotroph, such as a species ofPrototheca, including Prototheca moriformis or Prototheca zopfii. Thefats can also be produced in autotrophic algae or plants. Optionally,the cell is capable of using sucrose to produce oil and a recombinantinvertase gene may be introduced to allow metabolism of sucrose, asdescribed in PCT Publications WO2008/151149, WO2010/06032,WO2011/150410, WO2011/150411, and international patent applicationPCT/US12/23696. The invertase may be codon optimized and integrated intoa chromosome of the cell, as may all of the genes mentioned here.

In an embodiment, the natural fat has at least 30, 40, 50, 60, 70, 80,or 90% fat of the general structure [saturated fatty acid(sn-1)-unsaturated fatty acid (sn-2)-saturated fatty acid (sn-3)]. Thisis denoted below as Sat-Unsat-Sat fat. In a specific embodiment, thesaturated fatty acid in this structure is preferably stearate orpalmitate and the unsaturated fatty acid is preferably oleate. As aresult, the fat can form primarily β or β′ polymorphic crystals, or amixture of these, and have corresponding physical properties, includingthose desirable for use in foods or personal care products. For example,the fat can melt at mouth temperature for a food product or skintemperature for a cream, lotion or other personal care product (e.g., amelting temperature of 30 to 40, or 32 to 35° C.). Optionally, the fatscan have a 2 L or 3 L lamellar structure (e.g., as determined by X-raydiffraction analysis). Optionally, the fat can form this polymorphicform without tempering.

In a specific related embodiment, a natural fat triglyceride has a highconcentration of SOS (i.e. triglyceride with stearate at the terminalsn-1 and sn-3 positions, with oleate at the sn-2 position of theglycerol backbone). For example, the fat can have triglyceridescomprising at least 50, 60, 70, 80 or 90% SOS. In an embodiment, the fathas triglyceride of at least 80% SOS. Optionally, at least 50, 60, 70,80 or 90% of the sn-2 linked fatty acids are unsaturated fatty acids. Ina specific embodiment, at least 95% of the sn-2 linked fatty acids areunsaturated fatty acids. In addition, the SSS (tri-stearate) level canbe less than 20, 10 or 5% and/or the C20:0 fatty acid (arachidic acid)level may be less than 6%, and optionally greater than 1% (e.g., from 1to 5%). For example, in a specific embodiment, a natural fat produced bya recombinant cell has at least 70% SOS triglyceride with at least 80%sn-2 unsaturated fatty acyl moieties. In another specific embodiment, anatural fat produced by a recombinant cell has TAGs with at least 80%SOS triglyceride and with at least 95% sn-2 unsaturated fatty acylmoieties. In yet another specific embodiment, a natural fat produced bya recombinant cell has TAGs with at least 80% SOS, with at least 95%sn-2 unsaturated fatty acyl moieties, and between 1 to 6% C20 fattyacids.

In yet another specific embodiment, the sum of the percent stearate andpalmitate in the fatty acid profile of the natural fat is twice thepercentage of oleate, ±10, 20, 30 or 40% [e.g., (% P+% S)/%0=2.0±20%].Optionally, the sn-2 profile of this fat is at least 40%, and preferablyat least 50, 60, 70, or 80% oleate (at the sn-2 position). Alsooptionally, this fat may be at least 40, 50, 60, 70, 80, or 90% SOS.Optionally, the fat comprises between 1 to 6% C20 fatty acids.

In any of these embodiments, the high SatUnsatSat fat may tend to form(3′ polymorphic crystals. Unlike previously available plant fats likecocoa butter, the SatUnsatSat fat produced by the cell may form β′polymorphic crystals without tempering. In an embodiment, the polymorphforms upon heating to above melting temperature and cooling to less thatthe melting temperature for 3, 2, 1, or 0.5 hours. In a relatedembodiment, the polymorph forms upon heating to above 60° C. and coolingto 10° C. for 3, 2, 1, or 0.5 hours.

In various embodiments the fat forms polymorphs of the β form, β′ form,or both, when heated above melting temperature and the cooled to belowmelting temperature, and optionally proceeding to at least 50% ofpolymorphic equilibrium within 5, 4, 3, 2, 1, 0.5 hours or less whenheated to above melting temperature and then cooled at 10° C. The fatmay form β′ crystals at a rate faster than that of cocoa butter.

Optionally, any of these fats can have less than 2 mole %diacylglycerol, or less than 2 mole % mono and diacylglycerols, in sum.

In an embodiment, the fat may have a melting temperature of between30-60° C., 30-40° C., 32 to 37° C., 40 to 60° C. or 45 to 55° C. Inanother embodiment, the fat can have a solid fat content (SFC) of 40 to50%, 15 to 25%, or less than 15% at 20° C. and/or have an SFC of lessthan 15% at 35° C.

The cell used to make the fat may include recombinant nucleic acidsoperable to modify the saturate to unsaturate ratio of the fatty acidsin the cell triglyceride in order to favor the formation of SatUnsatSatfat. For example, a knock-out or knock-down of stearoyl-ACP desaturase(SAD) gene can be used to favor the formation of stearate over oleate orexpression of an exogenous mid-chain-preferring acyl-ACP thioesterasecan increase the levels mid-chain saturates. Alternately a gene encodinga SAD enzyme can be overexpressed to increase unsaturates.

In a specific embodiment, the cell has recombinant nucleic acidsoperable to elevate the level of stearate in the cell. As a result, theconcentration of SOS may be increased. Example 9 demonstrates that theregiospecific profile of the recombinant microbe is enriched for theSatUnsatSat triglycerides POP, POS, and SOS as a result ofoverexpressing a Brassica napus C18:0-preferring thioesterase. Anadditional way to increase the stearate of a cell is to decrease oleatelevels. For cells having high oleate levels (e.g., in excess of one halfthe stearate levels) one can also employ recombinant nucleic acids orclassical genetic mutations operable to decrease oleate levels. Forexample, the cell can have a knockout, knockdown, or mutation in one ormore FATA alleles, which encode an oleate liberating acyl-ACPthioesterase, and/or one or more alleles encoding a stearoyl ACPdesaturase (SAD). Example 35 describes the inhibition of SAD2 geneproduct expression using hairpin RNA to produce a fatty acid profile of37% stearate in Prototheca moriformis (UTEX 1435), whereas the wildtypestrain produced less than 4% stearate, a more than 9-fold improvement.Moreover, while the strains of Example 35 are engineered to reduce SADactivity, sufficient SAD activity remains to produce enough oleate tomake SOS, POP, and POS. See the TAG profiles of Example 47. In specificexamples, one of multiple SAD encoding alleles may be knocked out and/orone or more alleles are downregulated using inhibition techniques suchas antisense, RNAi, or siRNA, hairpin RNA or a combination thereof. Invarious embodiments, the cell can produce TAGs that have 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, or 90 to about 100% stearate. Inother embodiments, the cells can produce TAGs that are 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, or 90 to about 100% SOS. Optionally,or in addition to genetic modification, stearoyl ACP desaturase can beinhibited chemically; e.g., by addition of sterculic acid to the cellculture during oil production.

Surprisingly, knockout of a single FATA allele has been found toincrease the presence of C18 fatty acids produced in microalgae. Byknocking out one allele, or otherwise suppressing the activity of theFATA gene product (e.g., using hairpin RNA), while also suppressing theactivity of stearoyl-ACP desaturase (using techniques disclosed herein),stearate levels in the cell can be increased.

Another genetic modification to increase stearate levels includesincreasing a ketoacyl ACP synthase (KAS) activity in the cell so as toincrease the rate of stearate production. It has been found that inmicroalgae, increasing KASII activity is effective in increasing C18synthesis and particularly effective in elevating stearate levels incell triglyceride in combination with recombinant DNA effective indecreasing SAD activity. Recombinant nucleic acids operable to increaseKASII (e.g., an exogenous KasII gene) can be also be combined with aknockout or knockdown of a FatA gene, or with knockouts or knockdowns ofboth a FatA gene and a SAD gene).

Optionally, the cell can include an exogenous stearate liberatingacyl-ACP thioesterase, either as a sole modification or in combinationwith one or more other stearate-increasing genetic modifications. Forexample the cell may be engineered to overexpress an acyl-ACPthioesterase with preference for cleaving C18:0-ACPs. Example 9describes the expression of exogenous C18:0-preferring acyl-ACPthioesterases to increase stearate in the fatty acid profile of themicroalgae Prototheca moriformis (UTEX 1435) from about 3.7% to about30.4% (over 8-fold). Example 41 provides additional examples ofC18:0-preferring acyl-ACP thioesterases function to elevate C18:0 levelsin Prototheca. Introduction of the acyl-ACP thioesterase can be combinedwith a knockout or knockdown of one or more endogenous acyl-ACPthioesterase alleles. Introduction of the thioesterase can also becombined with overexpression of an elonagase (KCS) or beta-ketoacyl-CoAsynthase. In addition, one or more exogenous genes (e.g., encoding SADor KASII) can be regulated via an environmental condition (e.g., byplacement in operable linkage with a regulatable promoter). In aspecific example, pH and/or nitrogen level is used to regulate an amt03promoter. The environmental condition may then be modulated to tune thecell to produce the desired amount of stearate appearing in celltriglycerides (e.g., to twice the oleate concentration). As a result ofthese manipulations, the cell may exhibit an increase in stearate of atleast 5, 10, 15, or 20 fold.

As a further modification alone or in combination with the otherstearate increasing modifications, the cell can comprise recombinantnucleic acids operable to express an elongase or a beta-ketoacyl-CoAsynthase. For example, overexpression of a C18:0-preferring acyl-ACPthioesterases may be combined with overexpression of amidchain-extending elongase or KCS to increase the production ofstearate in the recombinant cell. One or more of the exogenous genes(e.g., ending a thioesterase, elongase, or KCS) can be regulated via anenvironmental condition (e.g., by placement in operable linkage with aregulatable promoter). In a specific example, pH and/or nitrogen levelis used to regulate an amt03 promoter. The environmental condition maythen be modulated to tune the cell to produce the desired amount ofstearate appearing in cell triglycerides (e.g., to twice the oleateconcentration). As a result of these manipulations, the cell may exhibitan increase in stearate of at least 5, 10, 15, or 20 fold. In additionto stearate, arachidic, behenic, lignoceric, and cerotic acids may alsobe produced.

In specific embodiments, due to the genetic manipulations of the cell toincrease stearate levels, the ratio of stearate to oleate in the oilproduced by the cell is 3:1±30% (i.e., in the range of 2.7:1 to 3.3:1),3:1±20% or 3:1±10%.

Alternately, the cell can be engineered to favor formation ofSatUnsatSat where Sat is palmitate or a mixture of palmitate andstearate. In this case introduction of an exogenous palmitate liberatingacyl-ACP thioesterase can promote palmitate formation. In thisembodiment, the cell can produce triglycerides, that are at least 30,40, 50, 60, 70, or 80% POP, or triglycerides in which the sum of POP,SOS, and POS is at least 30, 40, 50, 60, 70, 80, or 90% of celltriglycerides. In other related embodiments, the POS level is at least30, 40, 50, 60, 70, 80, or 90% of the triglycerides produced by thecell.

In a specific embodiment, the melting temperature of the oil is similarto that of cocoa butter (about 30-32° C.). The POP, POS and SOS levelscan approximate cocoa butter at about 16, 38, and 23% respectively. Forexample, POP can be 16%±20%, POS can be 38%±20%, and SOS can be 23%±20%.Or, POP can be 16%±15%, POS can be 38%±15%, an SOS can be 23%±15%. Or,POP can be 16%±10%, POS can be 38%±10%, an SOS can be 23%±10%.

As a result of the recombinant nucleic acids that increase stearate, aproportion of the fatty acid profile may be arachidic acid. For example,the fatty acid profile can be 0.01% to 5%, 0.1 to 4%, or 1 to 3%arachidic acid. Furthermore, the regiospecific profile may have 0.01% to4%, 0.05% to 3%, or 0.07% to 2% AOS, or may have 0.01% to 4%, 0.05% to3%, or 0.07% to 2% AOA. It is believed that AOS and AOA may reduceblooming and fat migration in confection comprising the fats of thepresent invention, among other potential benefits.

In addition to the manipulations designed to increase stearate and/orpalmitate, and to modify the SatUnsatSat levels, the levels ofpolyunsaturates may be suppressed, including as described above byreducing delta 12 fatty acid desaturase activity (e.g., as encoded by aFad gene) and optionally supplementing the growth medium or regulatingFAD expression. It has been discovered that, in microalgae (as evidencedby work in Prototheca strains), polyunsaturates are preferentially addedto the sn-2 position. Thus, to elevate the percent of triglycerides witholeate at the sn-2 position, production of linoleic acid by the cell maybe suppressed. The techniques described herein, in connection withhighly oxidatively stable oils, for inhibiting or ablating fatty aciddesaturase (FAD) genes or gene products may be applied with good effecttoward producing SatUnsatSat oils by reducing polyunsaturates at thesn-2 position. As an added benefit, such oils can have improvedoxidatively stability. As also described herein, the fats may beproduced in two stages with polyunsaturates supplied or produced by thecell in the first stage with a deficit of polyunsaturates during the fatproducing stage. The fat produced may have a fatty acid profile havingless than or equal to 15, 10, 7, 5, 4, 3, 2, 1, or 0.5% polyunsaturates.In a specific embodiment, the oil/fat produced by the cell has greaterthan 50% SatUnsatSat, and optionally greater than 50% SOS, yet has lessthan 3% polyunsaturates. Optionally, polyunsaturates can be approximatedby the sum of linoleic and linolenic acid area % in the fatty acidprofile.

In an embodiment, the natural fat is a Shea stearin substitute having65% to 95% SOS and optionally 0.001 to 5% SSS. In a related embodiment,the fat has 65% to 95% SOS, 0.001 to 5% SSS, and optionally 0.1 to 8%arachidic acid containing triglycerides. In another related embodiment,the fat has 65% to 95% SOS and the sum of SSS and SSO is less than 10%or less than 5%.

The cell's regiospecific preference can be learned using the analyticalmethod described below (Examples 1-2, 8). Despite balancing thesaturates and unsaturates as describe above, it is possible that thecell enzymes do not place the unsaturated fatty acid at the sn-2position. In this case, genetic manipulations can confer the desiredregiospecificity by (i) reducing the activity of endogenous sn-2specific acyl transferases (e.g., LPAAT) and/or (ii) introducing anexogenous LPAAT with the desired specificity (i.e., introduction ofoleate at sn-2). Where an exogenous LPAAT is introduced, preferably thegene encoding the LPAAT is integrated into a host chromosome and istargeted to the endoplasmic reticulum. In some cases, the host cell mayhave both specific and non-specific LPAAT alleles and suppressing theactivity of one of these alleles (e.g., with a gene knockout) willconfer the desired specificity. For example, genes encoding the LPAATsof SEQ ID NO: 78 and SEQ ID NO: 79 or an LPAAT comprising at least 90,95, 98, or 99% amino acid identity to either of these sequences, or afunctional fragment thereof, can be used to add oleate to the sn-2position in order to boost the levels of SatUnsatSat TAGs. The genes canhave at least 80, 85, 90, 95, 96, 97, 98, or 99% nucleotide identity toany of SEQ ID NOs: 80 to 85 or equivalent sequences by virtue of thedegeneracy of the genetic code. These genes can be manifest asrecombinant nucleic acid constructs, vectors, chromosomes or host cellscomprising these sequences or functional fragments thereof, which can befound by systematic deletion of nucleic acid from the sequences usingknown techniques. As a result of expression of the genes, the amount ofsat-unsat-sat TAGs such as SOS, POS, POP, or triglycerides with C8 toC16 fatty acids at the sn-2 position can be increased in a host cell.

In an embodiment, fats produced by cells according to the invention areused to produce a confection, candy coating, or other food product. As aresult, a food product like a chocolate or candy bar may have the “snap”(e.g., when broken) of a similar product produced using cocoa butter.The fat used may be in a beta polymorphic form or tend to a betapolymorphic form. In an embodiment, a method includes adding such a fatto a confection. Optionally, the fat can be a cocoa butter equivalentper EEC regulations, having greater than 65% SOS, less than 45%unsaturated fatty acid, less than 5% polyunsaturated fatty acids, lessthan 1% lauric acid, and less than 2% trans fatty acid. The fats canalso be used as cocoa butter extenders, improvers, replacers, oranti-blooming agents, or as Shea butter replacers, including in food andpersonal care products. High SOS fats produced using the cells andmethods disclosed here can be used in any application or formulationthat calls for Shea butter or Shea fraction. However, unlike Sheabutter, fats produced by the embodiments of the invention can have lowamounts of unsaponifiables; e.g. less than 7, 5, 3, or 2%unsaponifiables. In addition, Shea butter tends to degrade quickly dueto the presence of diacylglycerides whereas fats produced by theembodiments of the invention can have low amounts of diacylglycerides;e.g., less than 5, 4, 3, 2, 1, or 0.5% diacylglycerides.

In an embodiment of the invention there is a natural fat suitable as ashortening, and in particular, as a roll-in shortening. Thus, theshortening may be used to make pastries or other multi-laminate foods.The shortening can be produced using methods disclosed herein forproducing engineered organisms and especially heterotrophic microalgae.In an embodiment, the shortening has a melting temperature of between 40to 60° C. and preferably between 45-55° C. and can have a triglycerideprofile with 15 to 20% medium chain fatty acids (C8 to C14), 45-50% longchain saturated fatty acids (C16 and higher), and 30-35% unsaturatedfatty acids (preferably with more oleic than linoleic). The shorteningmay form (3′ polymorphic crystals, optionally without passing throughthe β polymorphic form. The shortening may be thixotropic. Theshortening may have a solid fat content of less than 15% at 35° C. In aspecific embodiment, there is a natural oil suitable as a roll-inshortening produced by a recombinant microalga, where the oil has ayield stress between 400 and 700 or 500 and 600 Pa and a storage modulusof greater than 1×10⁵ Pa or 1×10⁶ Pa. (see Example 46)

A structured solid-liquid fat system can be produced using thestructuring oils by blending them with an oil that is a liquid at roomtemperature (e.g., an oil high in tristearin or triolein). The blendedsystem may be suitable for use in a food spread, mayonnaise, dressing,shortening; i.e. by forming an oil-water-oil emulsion. The structuringfats according to the embodiments described here, and especially thosehigh in SOS, can be blended with other oils/fats to make a cocoa butterequivalent, replacer, or extender. For example, a natural fat havinggreater than 65% SOS can be blended with palm mid-fraction to make acocoa butter equivalent.

In general, such high Sat-Unsat-Sat fats or fat systems can be used in avariety of other products including whipped toppings, margarines,spreads, salad dressings, baked goods (e.g. breads, cookies, crackersmuffins, and pastries), cheeses, cream cheese, mayonnaise, etc.

In a specific embodiment, a Sat-Unsat-Sat fat described above is used toproduce a margarine, spread, or the like. For example, a margarine canbe made from the fat using any of the recipes or methods found in U.S.Pat. Nos. 7,118,773, 6,171,636, 4,447,462, 5,690,985, 5,888,575,5,972,412, 6,171,636, or international patent publications WO9108677A1.

In an embodiment, a fat comprises a natural (e.g., from microalgalcells) fat optionally blended with another fat and is useful forproducing a spread or margarine or other food product is produced by thegenetically engineered cell and has glycerides derived from fatty acidswhich comprises:

-   -   (a) at least 10 weight % of C18 to C24 saturated fatty acids,    -   (b) which comprise stearic and/or arachidic and/or behenic        and/or lignoceric acid and    -   (c) oleic and/or linoleic acid, while    -   (d) the ratio of saturated C18 acid/saturated        (C20+C22+C24)-acids≧1, preferably ≧5, more preferably ≧10, which        glycerides contain:    -   (e) ≦5 weight % of linolenic acid calculated on total fatty acid        weight    -   (f) ≦5 weight % of trans fatty acids calculated on total fatty        acid weight    -   (g) ≦75 weight %, preferably ≦60 weight % of oleic acid at the        sn-2 position: which glycerides contain calculated on total        glycerides weight

(h) ≧8 weight % HOH+HHO triglycerides

(i) ≦5 weight % of trisaturated triglycerides, and optionally one ormore of the following properties:

(j) a solid fat content of >10% at 10° C.

(k) a solid fat content≦15% at 35° C.,

-   -   (l) a solid fat content of >15% at 10° C. and a solid fat        content≦25% at 35° C.,    -   (m) the ratio of (HOH+HHO) and (HLH+HHL) triglycerides is >1,        and preferably >2,        -   where H stands for C18-C24 saturated fatty acid, O for oleic            acid, and L for linoleic acid.

Optionally, the solid content of the fat (% SFC) is 11 to 30 at 10° C.,4 to 15 at 20° C., 0.5 to 8 at 30° C., and 0 to 4 at 35° C. Alternately,the % SFC of the fat is 20 to 45 at 10° C., 14 to 25 at 20° C., 2 to 12at 30° C., and 0 to 5 at 35° C. In related embodiment, the % SFC of thefat is 30 to 60 at 10° C., 20 to 55 at 20° C., 5 to 35 at 30° C., and 0to 15 at 35° C. The C12-C16 fatty acid content can be ≦15 weight %. Thefat can have ≦5 weight % disaturated diglycerides.

In related embodiments there is a spread, margarine or other foodproduct made with the natural oil or natural oil blend. For example, thenatural fat can be used to make an edible W/O (water/oil) emulsionspread comprising 70-20 wt. % of an aqueous phase dispersed in 30-80 wt.% of a fat phase which fat phase is a mixture of 50-99 wt. % of avegetable triglyceride oil A and 1-50 wt. % of a structuringtriglyceride fat B, which fat consists of 5-100 wt. % of a hardstock fatC and up to 95 wt. % of a fat D, where at least 45 wt. % of thehardstock fat C triglycerides consist of SatOSat triglycerides and whereSat denotes a fatty acid residue with a saturated C18-C24 carbon chainand O denotes an oleic acid residue and with the proviso that anyhardstock fat C which has been obtained by fractionation, hydrogenation,esterification or interesterification of the fat is excluded. Thehardstock fat can be a natural fat produced by a cell according to themethods disclosed herein. Accordingly, the hardstock fat can be a fathaving a regiospecific profile having at least 50, 60, 70, 80, or 90%SOS. The W/O emulsion can be prepared to methods known in the artincluding in U.S. Pat. No. 7,118,773.

In related embodiment, the cell also expresses an endogenous hydrolyaseenzyme that produces ricinoleic acid. As a result, the oil (e.g., aliquid oil or structured fat) produced may be more easily emulsifiedinto a margarine, spread, or other food product or non-food product. Forexample, the oil produced may be emulsified using no added emulsifiersor using lower amounts of such emulsifiers. The U.S. patent applicationSer. No. 13/365,253 discloses methods for expressing such hydroxylasesin microalgae and other cells. In specific embodiments, a natural oilcomprises at least 1, 2, or 5% SRS, where S is stearate and R isricinoleic acid.

In an alternate embodiment, a natural oil that is a cocoa butter mimeticas described above can be fractionated to remove trisaturates (e.g.,tristearin and tripalmitin, SSP, and PPS). For example, it has beenfound that microalgae engineered to decrease SAD activity to increaseSOS concentration make an oil that can be fractionated to removetrisaturated. See Example 47. In specific embodiments, the meltingtemperature of the fractionated natural oil is similar to that of cocoabutter (about 30-32° C.). The POP, POS and SOS levels can approximatecocoa butter at about 16, 38, and 23% respectively. For example, POP canbe 16%±20%, POS can be 38%±20%, an SOS can be 23%±20%. Or, POP can be16%±15%, POS can be 38%±15%, an SOS can be 23%±15%. Or, POP can be16%±10%, POS can be 38%±10%, an SOS can be 23%±10%. In addition, thetristearin levels can be less than 5% of the triacylglycerides.

VIII. High Mid-Chain Oils

In an embodiment of the present invention, the cell has recombinantnucleic acids operable to elevate the level of midchain fatty acids(e.g., C8:0, C10:0, C12:0, C14:0, or C16:0 fatty acids) in the cell orin the oil of the cell. One way to increase the levels of midchain fattyacids in the cell or in the oil of the cell is to engineer a cell toexpress an exogenous acyl-ACP thioesterase that has activity towardsmidchain fatty acyl-ACP substrates (e.g., one encoded by a FatB gene),either as a sole modification or in combination with one or more othergenetic modifications. An additional genetic modification to increasethe level of midchain fatty acids in the cell or oil of the cell is theexpression of an exogenous lysophosphatidic acid acyltransferase geneencoding an active lysophosphatidic acid acyltransferase (LPAAT) thatcatalyzes the transfer of a mid-chain fatty-acyl group to the sn-2position of a substituted acylglyceroester. In a specific relatedembodiment, both an exogenous acyl-ACP thioesterase and LPAAT are stablyexpressed in the cell. In an embodiment, recombinant nucleic acids areintroduced into an oleaginous cell (and especially into a plastidicmicrobial cell) that cause expression of an exogenous mid-chain-specificthioesterase and an exogenous LPAAT that catalyzes the transfer of amid-chain fatty-acyl group to the sn-2 position of a substitutedacylglyceroester. As a result, the cell can be made to increase thepercent of a midchain fatty acid in the TAGs that it produces by 10, 2030, 40, 50, 60, 70, 80, 90-fold, or more. Introduction of the exogenousLPAAT can increase midchain fatty acids at the sn-2 position by 1.2,1.5, 1.7, 2, 3, 4 fold or more compared to introducing an exogenousmid-chain preferring acyl-ACP thioesterase alone. In an embodiment, themid-chain fatty acid is greater than 30, 40, 50 60, 70, 80, or 90% ofthe TAG fatty acids produced by the cell. In various embodiments, themid-chain fatty acid is lauric, myristic, or palmitic. Examples 3, 43,and 44 describe expression of plant LPAATs in microalgal cells withresulting alterations in fatty acid profiles. As in the examples, thecells can also express an exogenous acyl-ACP thioesterase (which canalso be from a plant) with a preference for a given fatty acyl-ACP chainlength. For example, a microalgal cell can comprise exogenous genesencoding a LPAAT and an acyl-ACP thioesterase that preferentially cleaveC8, C10, C12, C14, C8-C12, or C8-C10 fatty acids. In a specificembodiment, such a cell is capable of producing a natural oil with afatty acid profile comprising 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, or 90-99%, >20%, >30%, >40%, >50%, >60%, >70%, >80%or >90% C8, C10, C12, C14, C8-C12, or C8-C10 fatty acids. Other LPAATscan preferentially cleave C16 or C18 fatty acids (see Example 44).Further genetic manipulation of the fatty acid desaturase pathway (e.g.,as described infra) can increase the stability of the oils.

Any of these natural oils can be interesterified. Interesterificationcan, for example, be used to lower the melting temperature or pour-pointof the oil. In a specific embodiment, the natural oil comprises at least50% of the sum of caprylic and capric acids and may be interesterifiedto reduce the pour point and/or kinematic viscosity. Such an oil(natural or interesterified) can optionally be a high stability oilcomprising, for example, less than 2% polyunsaturated fatty acids.

Alternately, or in addition to expression of an exogenous LPAAT, thecell may comprise recombinant nucleic acids that are operable to expressan exogenous KASI or KASIV enzyme and optionally to decrease oreliminate the activity of a KASII, which is particularly advantageouswhen a mid-chain-preferring acyl-ACP thioesterase is expressed. Example37 describes the engineering of Prototheca cells to overexpress KASI orKASIV enzymes in conjunction with a mid-chain preferring acyl-ACPthioesterase to generate strains in which production of C10-C12 fattyacids is about 59% of total fatty acids. Mid-chain production can alsobe increased by suppressing the activity of KASI and/or KASII (e.g.,using a knockout or knockdown). Example 38 details the chromosomalknockout of different alleles of Prototheca moriformis (UTEX 1435) KASIin conjunction with overexpression of a mid-chain preferring acyl-ACPthioesterase to achieve fatty acid profiles that are about 76% or 84%C10-C14 fatty acids. Example 39 provides recombinant cells and oilscharacterized by elevated midchain fatty acids as a result of expressionof KASI RNA hairpin polynucleotides. In addition to any of thesemodifications, unsaturated or polyunsaturated fatty acid production canbe suppressed (e.g., by knockout or knockdown) of a SAD or FAD enzyme.

In a particular embodiment, a recombinant cell produces TAG having 40%lauric acid or more. In another related embodiment, a recombinant cellproduces TAG having a fatty acid profile of 40% or more of myristic,caprylic, capric, or palmitic acid. For example, an oleaginousrecombinant clorophyte cell can produce 40% lauric or myristic acid inan oil that makes up 40, 50, or 60% or more of the cell's dry weight.

In a specific embodiment, a recombinant cell comprises nucleic acidsoperable to express a product of an exogenous gene encoding alysophosphatidic acid acyltransferase that catalyzes the transfer of amid-chain fatty-acyl group to the sn-2 position of a substitutedacylglyceroester and nucleic acids operable to express a product of anacyl-ACP thioesterase exogenous gene encoding an active acyl-ACPthioesterase that catalyzes the cleavage of mid-chain fatty acids fromACP. As a result, in one embodiment, the oil produced can becharacterized by a fatty acid profile elevated in C10 and C12 fattyacids and reduced in C16, C18, and C18:1 fatty acids as a result of therecombinant nucleic acids. See Example 3, in which overexpression of aCuphea wrightii acyl-ACP thioesterase and a Cocos nucifera LPAAT geneincreased the percentage of C12 fatty acids from about 0.04% in theuntransformed cells to about 46% and increased the percentage of C10fatty acids from about 0.01% in the untransformed cells to about 11%. Inrelated embodiments, the increase in midchain fatty acid production isgreater than 70%, from 75-85%, from 70-90%, from 90-200%, from 200-300%,from 300-400%, from 400-500%, or greater than 500%.

Average chain length can also be reduced by overexpression of aC18-specific acyl-ACP thioesterase. Recombinant nucleic acids operableto overexpress a C18 or other acyl-ACP thioesterase may be used alone orin combination with the other constructs described here to furtherreduce average chain length. Among other uses, the oils produced can beused as cocoa-butter/milk fat substitute. See Example 45 and thediscussion of FIG. 17. In an embodiment, one of the above described highmid-chain producing cells is further engineered to produce a lowpolyunsaturated oil by knocking out or knocking down one or more fattyacyl desaturases, as described above in section IV. Accordingly, the oilproduced can have the high stability characteristic mentioned in thatsection or in corresponding Examples. In a specific embodiment, the cellproduces an oil comprising greater than 30% midchain fatty acids and 5%or less polyunsaturates. In a related embodiment, the cell produces anoil comprising greater than 40% midchain fatty acids and 4% or lesspolyunsaturates. In a further related embodiment, the cell produces anoil comprising greater than 50% midchain fatty acids and 3% or lesspolyunsaturates.

The high mid-chain oils or fatty acids derived from hydrolysis of theseoils may be particularly useful in food, fuel and oleochemicalapplications including the production of lubricants and surfactants. Forexample, fatty acids derived from the cells can be esterified, cracked,reduced to an aldehyde or alcohol, aminated, sulfated, sulfonated, orsubjected to other chemical process known in the art.

In some embodiments, the natural oil is interesterified and thekinematic viscosity of the interesterified natural oil is less than 30,20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 centiStokes at 40° C. In someembodiments, the kinematic viscosity is less than 3 centiStokes at 40°C. In some embodiments, the pour point of an interesterified natural oilis less than, 5° C., 0° C., −10° C., −12° C., −15° C., −20° C., −25° C.,−30° C., −35° C., −40° C., −45° C., or −50° C. In some embodiments, thepour point is less than −10° C. In some embodiments, the pour point isless than −20° C.

Example 53 describes the use of a plant FatB gene in algae to produceoils in microalgae with greater than 60% myristate. In an embodiment, agene encoding a protein having at least 90, 95, 96, 97, 98, or 99% aminoacid identity to SEQ ID NO:87 or SEQ ID NO:89 is used, optionally incombination with a mid-chain preferred LPAAT as described above.

IX. High Oleic/Palmitic Oil

In another embodiment, there is a high oleic oil with about 60% oleicacid, 25% palmitic acid and optionally 5% polyunsaturates or less. Thehigh oleic oil can be produced using the methods disclosed in U.S.patent application Ser. No. 13/365,253, which is incorporated byreference in relevant part. For example, the cell can have nucleic acidsoperable to suppress an acyl-ACP thioesterase (e.g., knockout orknockdown of a gene encoding FATA) while also expressing an gene thatincreases KASII activity. The cell can have further modifications toinhibit expression of delta 12 fatty acid desaturase, includingregulation of gene expression as described above. As a result, thepolyunsaturates can be less than or equal to 5, 4, 3, 2, or 1 area %.

X. Low Saturate Oil

In an embodiment, a natural oil is produced from a recombinant cell. Theoil produced has a fatty acid profile that has less that 4%, 3%, 2%, or1% (area %), saturated fatty acids. In a specific embodiment, the oilhas 0.1 to 3.5% saturated fatty acids. Certain of such oils can be usedto produce a food with negligible amounts of saturated fatty acids.Optionally, these oils can have fatty acid profiles comprising at least90% oleic acid or at least 90% oleic acid with at least 3%polyunsaturated fatty acids. In an embodiment, a natural oil produced bya recombinant cell comprises at least 90% oleic acid, at least 3% of thesum of linoleic and linolenic acid and has less than 3.5% saturatedfatty acids. In a related embodiment, a natural oil produced by arecombinant cell comprises at least 90% oleic acid, at least 3% of thesum of linoleic and linolenic acid and has less than 3.5% saturatedfatty acids, the majority of the saturated fatty acids being comprisedof chain length 10 to 16. These oils may be produced by recombinantoleaginous cells including but not limited to those described here andin U.S. patent application Ser. No. 13/365,253. For example,overexpression of a KASII enzyme in a cell with a highly active SAD canproduce a high oleic oil with less than or equal to 3.5% saturates.Optionally, an oleate-specific acyl-ACP thioesterase is alsooverexpressed and/or an endogenous thioesterase having a propensity tohydrolyze acyl chains of less than C18 knocked out or suppressed. Theoleate-specific acyl-ACP thioesterase may be a transgene with lowactivity toward ACP-palmitate and ACP-stearate so that the ratio ofoleic acid relative to the sum of palmitic acid and stearic acid in thefatty acid profile of the oil produced is greater than 3, 5, 7, or 10.Alternately, or in addition, a FATA gene may be knocked out or knockeddown, as in Example 36 below. A FATA gene may be knocked out or knockeddown and an exogenous KASII overexpressed. Another optional modificationis to increase KASI and/or KASIII activity, which can further suppressthe formation of shorter chain saturates. Optionally, one or moreacyltransferases (e.g., an LPAAT) having specificity for transferringunsaturated fatty acyl moieties to a substituted glycerol is alsooverexpressed and/or an endogenous acyltransferase is knocked out orattenuated. An additional optional modification is to increase theactivity of KCS enzymes having specificity for elongating unsaturatedfatty acids and/or an endogenous KCS having specificity for elongatingsaturated fatty acids is knocked out or attenuated. Optionally, oleateis increased at the expense of linoleate production by knockout orknockdown of a delta 12 fatty acid desaturase; e.g., using thetechniques of Section IV of this patent application. Optionally, theexogenous genes used can be plant genes; e.g., obtained from cDNAderived from mRNA found in oil seeds.

As described in Example 51, levels of saturated fats may also be reducedby introduction of an exogenous gene (e.g. a plant gene) thatdesaturates palmitic acid to palmitoleic acid. Examples of suitablegenes for use in the oleaginous cells are found in the plants, includingMacfadyena unguis (Cat's claw), Macadamia integrifolia (Macadamia nut)and Hippophae rhamnoides (sea buckthorn). Variant exogenous orendogenous SADs that desaturate palmitoyl-ACP can also be used and arefurther discussed in Example 51. Optionally, the PAD or SAD gene has atleast 95% amino acid sequence identity to the gene product described inExample 51. This modification can be used alone, or in combination witholeate-increasing modifications such as those described immediatelyabove, in section IX and in the Examples, including knockout orknockdown of one or more endogenous FATA alleles and/or overexpressionof KASII. In one embodiment, an oleaginous cell such as an oleaginousmicroalgae has a combination of (i) a FATA knockout or knockdown with(ii) expression of an exogenous PAD gene (this could also be a variantSAD with PAD activity, see Example 55) and/or a mutation in anendogenous SAD gene to give PAD activity. Such as cell may furthercomprise an overexpressed endogenous or exogenous KASII gene. Inaccordance with any of these embodiments of the invention, theoleaginous cell produces an oil having a fatty acid profile with 1-2,2-3, 3-4, 5-6, 7-8, 9-10, 10-15, 15-20, 20-30, 30-40, 40-60, 60-70,70-80, 80-90, or 90-100 area percent palmitoleic acid. In a specificembodiment, the cell produces greater than 50% oleic acid, greater than1% palmitoleic acid, and 3.5 area % or less of saturated fatty acids.

In addition to the above genetic modifications, the low saturate oil canbe a high-stability oil by virtue of low amounts of polyunsaturatedfatty acids. Methods and characterizations of high-stability,low-polyunsaturated oils are described in the section above entitled LowPolyunsaturated Oils, including method to reduce the activity ofendogenous Δ12 fatty acid desaturase. In a specific embodiment, an oilis produced by a oleaginous microbial cell having a type II fatty acidsynthetic pathway and has no more than 3.5% saturated fatty acids andalso has no more than 3% polyunsaturated fatty acids. In anotherspecific embodiment, the oil has no more than 3% saturated fatty acidsand also has no more than 2% polyunsaturated fatty acids. In anotherspecific embodiment, the oil has no more than 3% saturated fatty acidsand also has no more than 1% polyunsaturated fatty acids.

The low saturate and low saturate/high stability oil can be blended withless expensive oils to reach a targeted saturated fatty acid level atless expense. For example, an oil with 1% saturated fat can be blendedwith an oil having 7% saturated fat (e.g. high-oleic sunflower oil) togive an oil having 3-5% or less saturated fat.

Oils produced according to embodiments of the present invention can beused in the transportation fuel, oleochemical, and/or food and cosmeticindustries, among other applications. For example, transesterificationof lipids can yield long-chain fatty acid esters useful as biodiesel.Other enzymatic and chemical processes can be tailored to yield fattyacids, aldehydes, alcohols, alkanes, and alkenes. In some applications,renewable diesel, jet fuel, or other hydrocarbon compounds are produced.The present disclosure also provides methods of cultivating microalgaefor increased productivity and increased lipid yield, and/or for morecost-effective production of the compositions described herein. Themethods described here allow for the production of oils from plastidiccell cultures at large scale; e.g., 1000, 10,000, 100,000 liters ormore.

In an embodiment, an oil extracted from the cell has 3.5%, 3%, 2.5%, or2% saturated fat or less and is incorporated into a food product. Thefinished food product has 3.5, 3, 2.5, or 2% saturated fat or less.

XI. Cocoa Butter/Milk-Fat Blend Mimetics

In certain embodiments, the cell produces a natural oil that has atemperature-dependent solid fat content (“SFC-curve”) that approximatesa blend of cocoa butter and milk fat. Such oils may be used where thecocoa butter/milk fat blend could be used; for example, in chocolatesother confections, ice cream or other frozen desserts, pastries, ordough, including for quickbreads, or other baked goods. The oils mayinhibit blooming, enhance flavor, enhance texture, or reduce costs. In aspecific example, the natural oil approximates. Accordingly, anembodiment of the invention is using a natural oil from a recombinantmicroalgal cell to replace a cocoa butter/milfat blend in a recipe. In arelated embodiment,

FIG. 17 shows a plot of % solid fat content for various oils as follows(a) P. moriformis RBD oil without lipid pathway engineering, (b)Brazilian cocoa butter+25% milk fat, (c) three replicates of P.moriformis RBD oil from a strain expressing hairpin nucleic acids thatreduce levels of a SAD allele thus reducing oleic acid and increasingstearic acid in the TAG profile, (d) P. moriformis RBD oil from a strainoverexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, seeExample 45), (e) Malaysian cocoa butter+25% milk fat, and (f) Malaysiancocoa butter. The cocoa butter and cocoa butter milk fat values areliterature values (Bailey's Industrial Oils and Fat Products, 6^(th)ed.)

In an embodiment of the present invention, a natural oil that is similarin thermal properties to a 75% cocoa butter/25% milk fat blend isproduced by a microalgal or other cell described above. The cellcomprises recombinant nucleic acids operable to alter the fatty acidprofile of triglycerides produced by the cell so as that the oil has asolid fat content (e.g., as determined by NMR) of 38%±30% at 20° C.,32%±30% at 25° C., 17%±30% at 30° C., and less than 5%±30% at 35° C. Forthe sake of clarity, ±10% refers to percent of the percent SFC (e.g.,30% of 5% SFC is 1.5% SFC so the range is 3.5 to 6.5% SFC at 35° C.). Inrelated embodiments, the oil has a solid fat content (e.g., asdetermined by NMR) of 38%±20% at 20° C., 32%±20% at 25° C., 17%±20% at30° C., and less than 5%±20% at 35° C. or the oil has a solid fatcontent (e.g., as determined by NMR) of 38%±10% at 20° C., 32%±10% at25° C., 17%±10% at 30° C., and less than 5%±10% at 35° C.

XII. Minor Oil Components

The oils produced according to the above methods in some cases are madeusing a microalgal host cell. As described above, the microalga can be,without limitation, fall in the classification of Chlorophyta,Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It hasbeen found that microalgae of Trebouxiophyceae can be distinguished fromvegetable oils based on their sterol profiles. Oil produced by Chlorellaprotothecoides was found to produce sterols that appeared to bebrassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol,when detected by GC-MS. This method of detection generally does notdistinguish between the two possible stereoisomers at C-24 in theabsence of special conditions. However, sterols produced by green algae(e.g. Chlorophyta and specifically Chlorella) have a C24βstereochemistry (J. K. Volkman, Appl. Microbiotechnol. “Sterols inMicroorganisms”, 2003, 60:495-506). Thus, molecules detected ascampesterol, stigmasterol, and β-sitosterol, are actually their C24epimers 22,23-dihydrobrassicasterol, poriferasterol and clionasterol,respectively.

Stereoisomer Sterol (common name) Systematic name* 24-methylcholest-Campesterol (24R)-24-methylcholest- 5-en-3-ol 5-en-3β-ol 22,23-(24S)-24-methylcholest-5- dihydrobrassicasterol en-3β-ol (Δ5-ergostenol)24-ethylcholest- β-Sitosterol (24R)-24-ethylcholest- 5-en-3-ol5-en-3β-ol Clionasterol (24S)-24-ethylcholest-5- en-3β-ol5,22-cholestadien- Stigmasterol (24S)-24-ethylcholesta- 24-ethyl-3-ol5,22-dien-3β-ol Poriferasterol (24R)-24-ethylcholesta- 5,22-dien-3β-ol*In the nomenclature, 24α = 24(R) and 24β = 24(S) when side chain issaturated; 24α = 24(S) and 24β = 24(R) when side chain contains a Δ22double bond.Thus, the oils produced by the microalgae described above can bedistinguished from plant oils by the presence of sterols with C24βstereochemistry and the absence of C24α stereochemistry in the sterolspresent. For example, the oils produced may contain 22,23-dihydrobrassicasterol while lacking campesterol; containclionasterol, while lacking in β-sitosterol, and/or containporiferasterol while lacking stigmasterol. Alternately, or in addition,the oils may contain significant amounts of Δ⁷-poriferasterol.

In one embodiment, the oils provided herein are not vegetable oils.Vegetable oils are oils extracted from plants and plant seeds. Vegetableoils can be distinguished from the non-plant oils provided herein on thebasis of their oil content. A variety of methods for analyzing the oilcontent can be employed to determine the source of the oil or whetheradulteration of an oil provided herein with an oil of a different (e.g.plant) origin has occurred. The determination can be made on the basisof one or a combination of the analytical methods. These tests includebut are not limited to analysis of one or more of free fatty acids,fatty acid profile, total triacylglycerol content, diacylglycerolcontent, peroxide values, spectroscopic properties (e.g. UV absorption),sterol profile, sterol degradation products, antioxidants (e.g.tocopherols), pigments (e.g. chlorophyll), d13C values and sensoryanalysis (e.g. taste, odor, and mouth feel). Many such tests have beenstandardized for commercial oils such as the Codex Alimentariusstandards for edible fats and oils.

Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found inall eukaryotes. Animals exclusively make C27 sterols as they lack theability to further modify the C27 sterols to produce C28 and C29sterols. Plants, however, are able to synthesize C28 and C29 sterols,and C28/C29 plant sterols are often referred to as phytosterols. Thesterol profile of a given plant is high in C29 sterols, and the primarysterols in plants are typically the C29 sterols β-sitosterol andstigmasterol. In contrast, the sterol profile of non-plant organismscontains greater percentages of C27 and C28 sterols. For example, thesterols in fungi and in many microalgae are principally C28 sterols. Thesterol profile and particularly the striking predominance of C29 sterolsover C28 sterols in plants has been exploited for determining theproportion of plant and marine matter in soil samples (Huang, Wen-Yen,Meinschein W. G., “Sterols as ecological indicators”; Geochimica etCosmochimia Acta. Vol 43. pp 739-745).

Sterol profile analysis is a particularly well-known method fordetermining the biological source of organic matter. Campesterol,β-sitosterol, and stigmasterol are common plant sterols, withβ-sitosterol being a principle plant sterol. For example, β-sitosterolwas found to be in greatest abundance in an analysis of certain seedoils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74%in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Celland Molecular Biology 5:71-79, 2006).

Oil isolated from Prototheca moriformis strain UTEX1435 were separatelyclarified (CL), refined and bleached (RB), or refined, bleached anddeodorized (RBD) and were tested for sterol content according to theprocedure described in JAOCS vol. 60, no. 8, August 1983. Results of theanalysis are shown below (units in mg/100 g):

Refined, Refined & bleached, & Sterol Crude Clarified bleacheddeodorized 1 Ergosterol 384   398   293   302    (56%)  (55%)  (50%) (50%) 2 5,22-cholestadien-24-methyl-3-ol 14.6 18.8 14   15.2(Brassicasterol) (2.1%) (2.6%) (2.4%) (2.5%) 324-methylcholest-5-en-3-ol 10.7 11.9 10.9 10.8 (Campesterol or 22,23-(1.6%) (1.6%) (1.8%) (1.8%) dihydrobrassicasterol) 45,22-cholestadien-24-ethyl-3-ol 57.7 59.2 46.8 49.9 (Stigmasterol orporiferasterol) (8.4%) (8.2%) (7.9%) (8.3%) 5 24-ethylcholest-5-en-3-ol 9.64  9.92  9.26 10.2 (β-Sitosterol or clionasterol) (1.4%) (1.4%)(1.6%) (1.7%) 6 Other sterols 209   221   216   213   Total sterols685.64 718.82 589.96 601.1 

These results show three striking features. First, ergosterol was foundto be the most abundant of all the sterols, accounting for about 50% ormore of the total sterols. The amount of ergosterol is greater than thatof campesterol, β-sitosterol, and stigamsterol combined. Ergosterol issteroid commonly found in fungus and not commonly found in plants, andits presence particularly in significant amounts serves as a usefulmarker for non-plant oils. Secondly, the oil was found to containbrassicasterol. With the exception of rapeseed oil, brassicasterol isnot commonly found in plant based oils. Thirdly, less than 2%β-sitosterol (or clionasterol) was found to be present. β-sitosterol isa prominent plant sterol not commonly found in microalgae, and itspresence particularly in significant amounts serves as a useful markerfor oils of plant origin. As the specific stereoisomer of24-ethylcholest-5-en-3-ol was not confirmed, this sterol was most likelyclionosterol and not β-sitosterol in view of the current understandingof the sterols of the green algae (J. K. Volkman, Appl. Microbiotechnol.“Sterols in Microorganisms”, 2003, 60:495-506). In summary, Protothecamoriformis strain UTEX1435 has been found to contain both significantamounts of ergosterol and only trace amounts of24-ethylcholest-5-en-3-ol (as β-sitosterol or clionasterol) as apercentage of total sterol content. Accordingly, the ratio ofergosterol:β-sitosterol (or clionasterol) and/or in combination with thepresence of brassicasterol can be used to distinguish this oil fromplant oils.

In some embodiments, the oil content of an oil provided herein contains,as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%,2%, or 1% β-sitosterol and/or clionasterol.

In some embodiments, the oil is free from one or more of β-sitosterol,campesterol, or stigmasterol. In some embodiments the oil is free fromβ-sitosterol. In some embodiments the oil is free from campesterol. Insome embodiments the oil is free from stigmasterol.

In some embodiments, the oil content of an oil provided hereincomprises, as a percentage of total sterols, less than 20%, 15%, 10%,5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments,the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, theoil content of an oil provided herein comprises, as a percentage oftotal sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%clionasterol.

In some embodiments, the oil content of an oil provided herein contains,as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%,2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the24-methylcholest-5-en-3-ol is 22,23-dihydrobrassicasterol. In someembodiments, the oil content of an oil provided herein comprises, as apercentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, or 10% 22,23-dihydrobrassicasterol.

In some embodiments, the oil content of an oil provided herein contains,as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%,2%, or 1% 5,22-cholestadien-24-ethyl-3-ol. In some embodiments, the5,22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments,the oil content of an oil provided herein comprises, as a percentage oftotal sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%poriferasterol.

In some embodiments, the oil content of an oil provided herein containsergosterol or brassicasterol or a combination of the two. In someembodiments, the oil content contains, as a percentage of total sterols,at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%ergosterol. In some embodiments, the oil content contains, as apercentage of total sterols, at least 25% ergosterol. In someembodiments, the oil content contains, as a percentage of total sterols,at least 40% ergosterol. In some embodiments, the oil content contains,as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%,45%, 50%, 55%, 60%, or 65% of a combination of ergosterol andbrassicasterol.

In some embodiments, the oil content contains, as a percentage of totalsterols, at least 1%, 2%, 3%, 4% or 5% brassicasterol. In someembodiments, the oil content contains, as a percentage of total sterolsless than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.

In some embodiments the ratio of ergosterol to brassicasterol is atleast 5:1, 10:1, 15:1, or 20:1.

In some embodiments, the oil content contains, as a percentage of totalsterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%β-sitosterol. In some embodiments, the oil content contains, as apercentage of total sterols, at least 25% ergosterol and less than 5%β-sitosterol. In some embodiments, the oil content further comprisesbrassicasterol.

In some embodiments the primary sterols in the microalgal oils providedherein are sterols other than β-sitosterol and stigmasterol. In someembodiments of the microalgal oils, C29 sterols make up less than 50%,40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.

In some embodiments the microalgal oils provided herein contain C28sterols in excess of C29 sterols. In some embodiments of the microalgaloils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95%by weight of the total sterol content. In some embodiments the C28sterol is ergosterol. In some embodiments the C28 sterol isbrassicasterol.

XIII. Fuels and Chemicals

The oils discussed above alone or in combination are useful in theproduction of foods, fuels and chemicals (including plastics, foams,films, etc). The oils, triglycerides, fatty acids from the oils may besubjected to C—H activation, hydroamino methylation,methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation,methylation, dimerization, thiolation, metathesis, hydro-alkylation,lactonization, or other chemical processes.

The oils can be converted to alkanes (e.g., renewable diesel) or esters(e.g., methyl or ethyl esters for biodisesel produced bytransesterification). The alkanes or esters may be used as fuel, assolvents or lubricants, or as a chemical feedstock. Methods forproduction of renewable diesel and biodiesel are well established in theart. See, for example, WO2011/150411.

In a specific embodiment of the present invention, a high-oleic orhigh-oleic-high stability oil described above is esterified. Forexample, the oils can be transesterified with methanol to an oil that isrich in methyl oleate. As described in Example 49, such formulationshave been found to compare favorably with methyl oleate from soybeanoil.

In another specific example, the oil is converted to C36 diacids orproducts of C36 diacids. Fatty acids produced from the oil can bepolymerized to give a composition rich in C36 dimer acids. In a specificexample, high-oleic oil is split to give a high-oleic fatty acidmaterial which is polymerized to give a composition rich in C36-dimeracids. Optionally, the oil is high oleic high stability oil (e.g.,greater than 60% oleic acid with less than 3% polyunsaturates, greaterthan 70% oleic acid with less than 2% polyunsaturates, or greater than80% oleic acid with less than 1% polyunsaturates). It is believed thatusing a high oleic, high stability, starting material will give loweramounts of cyclic products, which may be desirable in some cases. Afterhydrolyzing the oil, one obtains a high concentration of oleic acid. Inthe process of making dimer acids, a high oleic acid stream will convertto a “cleaner” C36 dimer acid and not produce trimers acids (C54) andother more complex cyclic by-products which are obtained due to presenceof C18:2 and C18:3 acids. For example, the oil can be hydrolyzed tofatty acids and the fatty acids purified and dimerized at 250° C. in thepresence of montmorillonite clay. See SRI Natural Fatty Acid, March2009. A product rich in C36 dimers of oleic acid is recovered.

Further, the C36 dimer acids can be esterified and hydrogenated to givediols. The diols can be polymerized by catalytic dehydration. Polymerscan also be produced by transesterification of dimerdiols with dimethylcarbonate.

For the production of fuel in accordance with the methods of theinvention lipids produced by cells of the invention are harvested, orotherwise collected, by any convenient means. Lipids can be isolated bywhole cell extraction. The cells are first disrupted, and thenintracellular and cell membrane/cell wall-associated lipids as well asextracellular hydrocarbons can be separated from the cell mass, such asby use of centrifugation. Intracellular lipids produced in oleaginouscells are, in some embodiments, extracted after lysing the cells. Onceextracted, the lipids are further refined to produce oils, fuels, oroleochemicals.

Various methods are available for separating lipids from cellularlysates. For example, lipids and lipid derivatives such as fattyaldehydes, fatty alcohols, and hydrocarbons such as alkanes can beextracted with a hydrophobic solvent such as hexane (see Frenz et al.1989, Enzyme Microb. Technol., 11:717). Lipids and lipid derivatives canalso be extracted using liquefaction (see for example Sawayama et al.1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, BiomassBioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al.1995, Fuel 74(12):1735-1738); and supercritical CO₂ extraction (see forexample Mendes et al. 2003, Inorganica Chimica Acta 356:328-334). Miaoand Wu describe a protocol of the recovery of microalgal lipid from aculture of Chlorella protothecoides in which the cells were harvested bycentrifugation, washed with distilled water and dried by freeze drying.The resulting cell powder was pulverized in a mortar and then extractedwith n-hexane. Miao and Wu, Biosource Technology (2006) 97:841-846.

Lipids and lipid derivatives can be recovered by extraction with anorganic solvent. In some cases, the preferred organic solvent is hexane.Typically, the organic solvent is added directly to the lysate withoutprior separation of the lysate components. In one embodiment, the lysategenerated by one or more of the methods described above is contactedwith an organic solvent for a period of time sufficient to allow thelipid and/or hydrocarbon components to form a solution with the organicsolvent. In some cases, the solution can then be further refined torecover specific desired lipid or hydrocarbon components. Hexaneextraction methods are well known in the art.

Lipids produced by cells in vivo, or enzymatically modified in vitro, asdescribed herein can be optionally further processed by conventionalmeans. The processing can include “cracking” to reduce the size, andthus increase the hydrogen:carbon ratio, of hydrocarbon molecules.Catalytic and thermal cracking methods are routinely used in hydrocarbonand triglyceride oil processing. Catalytic methods involve the use of acatalyst, such as a solid acid catalyst. The catalyst can besilica-alumina or a zeolite, which result in the heterolytic, orasymmetric, breakage of a carbon-carbon bond to result in a carbocationand a hydride anion. These reactive intermediates then undergo eitherrearrangement or hydride transfer with another hydrocarbon. Thereactions can thus regenerate the intermediates to result in aself-propagating chain mechanism. Hydrocarbons can also be processed toreduce, optionally to zero, the number of carbon-carbon double, ortriple, bonds therein. Hydrocarbons can also be processed to remove oreliminate a ring or cyclic structure therein. Hydrocarbons can also beprocessed to increase the hydrogen:carbon ratio. This can include theaddition of hydrogen (“hydrogenation”) and/or the “cracking” ofhydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described below inthis Section.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manufacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is an urgent need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass or otherwise that meets the appropriateASTM specification can be defined as diesel fuel (ASTM D975), jet fuel(ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTMD6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear. Typically, biodiesel comprises C14-C18 alkyl esters.Various processes convert biomass or a lipid produced and isolated asdescribed herein to diesel fuels. A preferred method to producebiodiesel is by transesterification of a lipid as described herein. Apreferred alkyl ester for use as biodiesel is a methyl ester or ethylester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B100.

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel. The lipid profile of the biodiesel isusually highly similar to the lipid profile of the feedstock oil.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases. In a base-catalyzed transesterification process, thetriacylglycerides are reacted with an alcohol, such as methanol orethanol, in the presence of an alkaline catalyst, typically potassiumhydroxide. This reaction forms methyl or ethyl esters and glycerin(glycerol) as a byproduct.

Transesterification has also been carried out, as discussed above, usingan enzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80° C., and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1. Lipasessuitable for use in transesterification include, but are not limited to,those listed in Table 9. Other examples of lipases useful fortransesterification are found in, e.g., U.S. Pat. Nos. 4,798,793;4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipasesinclude, but are not limited to, lipases produced by microorganisms ofRhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida,and Humicola and pancreas lipase.

Subsequent processes may also be used if the biodiesel will be used inparticularly cold temperatures. Such processes include winterization andfractionation. Both processes are designed to improve the cold flow andwinter performance of the fuel by lowering the cloud point (thetemperature at which the biodiesel starts to crystallize). There areseveral approaches to winterizing biodiesel. One approach is to blendthe biodiesel with petroleum diesel. Another approach is to useadditives that can lower the cloud point of biodiesel. Another approachis to remove saturated methyl esters indiscriminately by mixing inadditives and allowing for the crystallization of saturates and thenfiltering out the crystals. Fractionation selectively separates methylesters into individual components or fractions, allowing for the removalor inclusion of specific methyl esters. Fractionation methods includeurea fractionation, solvent fractionation and thermal distillation.

Another valuable fuel provided by the methods of the present inventionis renewable diesel, which comprises alkanes, such as C10:0, C12:0,C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel.High quality renewable diesel conforms to the ASTM D975 standard. Thelipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In one embodiment, the method for producing renewable diesel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing the microorganism to produce a lysate, (c) isolatinglipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696. Some suitable methods may include mechanicalpressing and centrifuging.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet an ASTM D975standard while leaving components that are heavier than desired formeeting the D975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

In one embodiment of the method for producing renewable diesel, treatingthe lipid to produce an alkane is performed by hydrotreating of thelipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300° to 660°F., with pressure sufficient to keep the water primarily as a liquid,100 to 170 standard atmosphere (atm). Reaction times are on the order of15 to 30 minutes. After the reaction is completed, the organics areseparated from the water. Thereby a distillate suitable for diesel isproduced.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. No. 5,475,160(hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

One suitable method for the hydrogenation of triglycerides includespreparing an aqueous solution of copper, zinc, magnesium and lanthanumsalts and another solution of alkali metal or preferably, ammoniumcarbonate. The two solutions may be heated to a temperature of about 20°C. to about 85° C. and metered together into a precipitation containerat rates such that the pH in the precipitation container is maintainedbetween 5.5 and 7.5 in order to form a catalyst. Additional water may beused either initially in the precipitation container or addedconcurrently with the salt solution and precipitation solution. Theresulting precipitate may then be thoroughly washed, dried, calcined atabout 300° C. and activated in hydrogen at temperatures ranging fromabout 100° C. to about 400° C. One or more triglycerides may then becontacted and reacted with hydrogen in the presence of theabove-described catalyst in a reactor. The reactor may be a trickle bedreactor, fixed bed gas-solid reactor, packed bubble column reactor,continuously stirred tank reactor, a slurry phase reactor, or any othersuitable reactor type known in the art. The process may be carried outeither batchwise or in continuous fashion. Reaction temperatures aretypically in the range of from about 170° C. to about 250° C. whilereaction pressures are typically in the range of from about 300 psig toabout 2000 psig. Moreover, the molar ratio of hydrogen to triglyceridein the process of the present invention is typically in the range offrom about 20:1 to about 700:1. The process is typically carried out ata weight hourly space velocity (WHSV) in the range of from about 0.1hr⁻¹ to about 5 hr⁻¹. One skilled in the art will recognize that thetime period required for reaction will vary according to the temperatureused, the molar ratio of hydrogen to triglyceride, and the partialpressure of hydrogen. The products produced by the such hydrogenationprocesses include fatty alcohols, glycerol, traces of paraffins andunreacted triglycerides. These products are typically separated byconventional means such as, for example, distillation, extraction,filtration, crystallization, and the like.

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes. Solidcatalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Suitable methods for the deoxygenation of an oil includes heating an oilto a temperature in the range of from about 350° F. to about 550° F. andcontinuously contacting the heated oil with nitrogen under at leastpressure ranging from about atmospheric to above for at least about 5minutes.

Suitable methods for isomerization include using alkali isomerizationand other oil isomerization known in the art.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the triglyceride feed. The triglyceride molecule isreduced to four hydrocarbon molecules under hydroprocessing conditions:a propane molecule and three heavier hydrocarbon molecules, typically inthe C8 to C18 range.

Thus, in one embodiment, the product of one or more chemical reaction(s)performed on lipid compositions of the invention is an alkane mixturethat comprises ASTM D975 renewable diesel. Production of hydrocarbons bymicroorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol(2005) 66: 486-496 and A Look Back at the U.S. Department of Energy'sAquatic Species Program: Biodiesel from Algae, NREL/TP-580-24190, JohnSheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).

The distillation properties of a diesel fuel is described in terms ofT10-T90 (temperature at 10% and 90%, respectively, volume distilled).The T10-T90 of the material produced in Example 13 was 57.9° C. Methodsof hydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10-T90 ranges, such as 20, 25,30, 35, 40, 45, 50, 60 and 65° C. using triglyceride oils producedaccording to the methods disclosed herein.

Methods of hydrotreating, isomerization, and other covalent modificationof oils disclosed herein, as well as methods of distillation andfractionation (such as cold filtration) disclosed herein, can beemployed to generate renewable diesel compositions with other T10values, such as T10 between 180 and 295, between 190 and 270, between210 and 250, between 225 and 245, and at least 290.

Methods of hydrotreating, isomerization, and other covalent modificationof oils disclosed herein, as well as methods of distillation andfractionation (such as cold filtration) disclosed herein can be employedto generate renewable diesel compositions with certain T90 values, suchas T90 between 280 and 380, between 290 and 360, between 300 and 350,between 310 and 340, and at least 290.

The FBP of the material produced in Example 13 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other FBP values, such as FBP between290 and 400, between 300 and 385, between 310 and 370, between 315 and360, and at least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 1%-5%, preferably at least 4%, C8-C14; (b) at least 0.25%-1%,preferably at least 0.3%, C8; (c) at least 1%-5%, preferably at least2%, C10; (d) at least 1%-5%, preferably at least 2%, C12; and (3) atleast 20%-40%, preferably at least 30% C8-C14.

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

The present invention also provides methods to produce jet fuel. Jetfuel is clear to straw colored. The most common fuel is anunleaded/paraffin oil-based fuel classified as Aeroplane A-1, which isproduced to an internationally standardized set of specifications. Jetfuel is a mixture of a large number of different hydrocarbons, possiblyas many as a thousand or more. The range of their sizes (molecularweights or carbon numbers) is restricted by the requirements for theproduct, for example, freezing point or smoke point. Kerosone-typeAeroplane fuel (including Jet A and Jet A-1) has a carbon numberdistribution between about 8 and 16 carbon numbers. Wide-cut ornaphtha-type Aeroplane fuel (including Jet B) typically has a carbonnumber distribution between about 5 and 15 carbons.

In one embodiment of the invention, a jet fuel is produced by blendingalgal fuels with existing jet fuel. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. The lipidsproduced by the method of the present invention can be converted toolefins. The process involves flowing the lipids produced through an FCCzone and collecting a product stream comprised of olefins, which isuseful as a jet fuel. The lipids produced are contacted with a crackingcatalyst at cracking conditions to provide a product stream comprisingolefins and hydrocarbons useful as jet fuel.

In one embodiment, the method for producing jet fuel comprises (a)cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced. In one embodiment of themethod for producing a jet fuel, the lipid composition can be flowedthrough a fluid catalytic cracking zone, which, in one embodiment, maycomprise contacting the lipid composition with a cracking catalyst atcracking conditions to provide a product stream comprising C2-05olefins.

In certain embodiments of this method, it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C2-05 olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

Suitable FCC catalysts generally comprise at least two components thatmay or may not be on the same matrix. In some embodiments, both twocomponents may be circulated throughout the entire reaction vessel. Thefirst component generally includes any of the well-known catalysts thatare used in the art of fluidized catalytic cracking, such as an activeamorphous clay-type catalyst and/or a high activity, crystallinemolecular sieve. Molecular sieve catalysts may be preferred overamorphous catalysts because of their much-improved selectivity todesired products. In some preferred embodiments, zeolites may be used asthe molecular sieve in the FCC processes. Preferably, the first catalystcomponent comprises a large pore zeolite, such as a Y-type zeolite, anactive alumina material, a binder material, comprising either silica oralumina and an inert filler such as kaolin.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149° C. to about 316° C. (300° F.to 600° F.). The catalyst is flowed from a blending vessel to the riserwhere it contacts the lipid composition for a time of abort 2 seconds orless.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst to lipidcomposition ratio which can vary from about 5 to about 20 and preferablyfrom about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700° C. (1292° F.) to about 760° C. (1400° F.) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO). HDO means removal of oxygen bymeans of hydrogen, that is, oxygen is removed while breaking thestructure of the material. Olefinic double bonds are hydrogenated andany sulfur and nitrogen compounds are removed. Sulfur removal is calledhydrodesulphurization (HDS). Pretreatment and purity of the rawmaterials (lipid composition or the lipids) contribute to the servicelife of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerization reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulfur compounds, and lighthydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerization is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step, i.e. HDO step, of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle. In the HDO step, thepressure varies between 20 and 150 bar, preferably between 50 and 100bar, and the temperature varies between 200 and 500° C., preferably inthe range of 300-400° C. In the HDO step, known hydrogenation catalystscontaining metals from Group VII and/or VIB of the Periodic System maybe used. Preferably, the hydrogenation catalysts are supported Pd, Pt,Ni, NiMo or a CoMo catalysts, the support being alumina and/or silica.Typically, NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50-400° C. and at hydrogen pressures of 1-200 bar,preferably at a temperature between 150 and 250° C. and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulfurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapor or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner. In theisomerization step, the pressure varies in the range of 20-150 bar,preferably in the range of 20-100 bar, the temperature being between 200and 500° C., preferably between 300 and 400° C. In the isomerizationstep, isomerization catalysts known in the art may be used. Suitableisomerization catalysts contain molecular sieve and/or a metal fromGroup VII and/or a carrier. Preferably, the isomerization catalystcontains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pdor Ni and Al₂O₃ or SiO₂. Typical isomerization catalysts are, forexample, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂. The isomerization step and the HDO step may be carriedout in the same pressure vessel or in separate pressure vessels.Optional prehydrogenation may be carried out in a separate pressurevessel or in the same pressure vessel as the HDO and isomerizationsteps.

Thus, in one embodiment, the product of one or more chemical reactionsis an alkane mixture that comprises HRJ-5. In another embodiment, theproduct of the one or more chemical reactions is an alkane mixture thatcomprises ASTM D1655 jet fuel. In some embodiments, the compositioncomforming to the specification of ASTM 1655 jet fuel has a sulfurcontent that is less than 10 ppm. In other embodiments, the compositionconforming to the specification of ASTM 1655 jet fuel has a T10 value ofthe distillation curve of less than 205° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afinal boiling point (FBP) of less than 300° C. In another embodiment,the composition conforming to the specification of ASTM 1655 jet fuelhas a flash point of at least 38° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has adensity between 775 K/M³ and 840 K/M³. In yet another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afreezing point that is below −47° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anet Heat of Combustion that is at least 42.8 MJ/K. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a hydrogen content that is at least 13.4 mass %. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a thermal stability, as tested by quantitative gravimetricJFTOT at 260° C., which is below 3 mm of Hg. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anexistent gum that is below 7 mg/dl.

Thus, the present invention discloses a variety of methods in whichchemical modification of microalgal lipid is undertaken to yieldproducts useful in a variety of industrial and other applications.Examples of processes for modifying oil produced by the methodsdisclosed herein include, but are not limited to, hydrolysis of the oil,hydroprocessing of the oil, and esterification of the oil. Otherchemical modification of microalgal lipid include, without limitation,epoxidation, oxidation, hydrolysis, sulfations, sulfonation,ethoxylation, propoxylation, amidation, and saponification. Themodification of the microalgal oil produces basic oleochemicals that canbe further modified into selected derivative oleochemicals for a desiredfunction. In a manner similar to that described above with reference tofuel producing processes, these chemical modifications can also beperformed on oils generated from the microbial cultures describedherein. Examples of basic oleochemicals include, but are not limited to,soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogencompounds including fatty amides, fatty acid methyl esters, andglycerol. Examples of derivative oleochemicals include, but are notlimited to, fatty nitriles, esters, dimer acids, quats, surfactants,fatty alkanolamides, fatty alcohol sulfates, resins, emulsifiers, fattyalcohols, olefins, drilling muds, polyols, polyurethanes, polyacrylates,rubber, candles, cosmetics, metallic soaps, soaps, alpha-sulphonatedmethyl esters, fatty alcohol sulfates, fatty alcohol ethoxylates, fattyalcohol ether sulfates, imidazolines, surfactants, detergents, esters,quats, ozonolysis products, fatty amines, fatty alkanolamides,ethoxysulfates, monoglycerides, diglycerides, triglycerides (includingmedium chain triglycerides), lubricants, hydraulic fluids, greases,dielectric fluids, mold release agents, metal working fluids, heattransfer fluids, other functional fluids, industrial chemicals (e.g.,cleaners, textile processing aids, plasticizers, stabilizers,additives), surface coatings, paints and lacquers, electrical wiringinsulation, and higher alkanes. Rubber compositions comprising microbialoils (e.g., microalgal oil) can be used in, e.g., the manufacture oftire treads and/or tires. Elastomers and additives for use inpreparation of the rubber compositions disclosed herein are described inU.S. Pat. No. 7,253,225, U.S. Pat. No. 7,335,692, U.S. Pat. No.8,324,310, and US 2013/0096248, which are hereby incorporated byreference in their entirety.

Hydrolysis of the fatty acid constituents from the glycerolipidsproduced by the methods of the invention yields free fatty acids thatcan be derivatized to produce other useful chemicals. Hydrolysis occursin the presence of water and a catalyst which may be either an acid or abase. The liberated free fatty acids can be derivatized to yield avariety of products, as reported in the following: U.S. Pat. No.5,304,664 (Highly sulfated fatty acids); U.S. Pat. No. 7,262,158(Cleansing compositions); U.S. Pat. No. 7,115,173 (Fabric softenercompositions); U.S. Pat. No. 6,342,208 (Emulsions for treating skin);U.S. Pat. No. 7,264,886 (Water repellant compositions); U.S. Pat. No.6,924,333 (Paint additives); U.S. Pat. No. 6,596,768 (Lipid-enrichedruminant feedstock); and U.S. Pat. No. 6,380,410 (Surfactants fordetergents and cleaners).

In some methods, the first step of chemical modification may behydroprocessing to saturate double bonds, followed by deoxygenation atelevated temperature in the presence of hydrogen and a catalyst. Inother methods, hydrogenation and deoxygenation may occur in the samereaction. In still other methods deoxygenation occurs beforehydrogenation. Isomerization may then be optionally performed, also inthe presence of hydrogen and a catalyst. Finally, gases and naphthacomponents can be removed if desired. For example, see U.S. Pat. No.5,475,160 (hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

In some embodiments of the invention, the triglyceride oils arepartially or completely deoxygenated. The deoxygenation reactions formdesired products, including, but not limited to, fatty acids, fattyalcohols, polyols, ketones, and aldehydes. In general, without beinglimited by any particular theory, the deoxygenation reactions involve acombination of various different reaction pathways, including withoutlimitation: hydrogenolysis, hydrogenation, consecutivehydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation,and combined hydrogenation-hydrogenolysis reactions, resulting in atleast the partial removal of oxygen from the fatty acid or fatty acidester to produce reaction products, such as fatty alcohols, that can beeasily converted to the desired chemicals by further processing. Forexample, in one embodiment, a fatty alcohol may be converted to olefinsthrough FCC reaction or to higher alkanes through a condensationreaction.

One such chemical modification is hydrogenation, which is the additionof hydrogen to double bonds in the fatty acid constituents ofglycerolipids or of free fatty acids. The hydrogenation process permitsthe transformation of liquid oils into semi-solid or solid fats, whichmay be more suitable for specific applications.

Hydrogenation of oil produced by the methods described herein can beperformed in conjunction with one or more of the methods and/ormaterials provided herein, as reported in the following: U.S. Pat. No.7,288,278 (Food additives or medicaments); U.S. Pat. No. 5,346,724(Lubrication products); U.S. Pat. No. 5,475,160 (Fatty alcohols); U.S.Pat. No. 5,091,116 (Edible oils); U.S. Pat. No. 6,808,737 (Structuralfats for margarine and spreads); U.S. Pat. No. 5,298,637(Reduced-calorie fat substitutes); U.S. Pat. No. 6,391,815(Hydrogenation catalyst and sulfur adsorbent); U.S. Pat. Nos. 5,233,099and 5,233,100 (Fatty alcohols); U.S. Pat. No. 4,584,139 (Hydrogenationcatalysts); U.S. Pat. No. 6,057,375 (Foam suppressing agents); and U.S.Pat. No. 7,118,773 (Edible emulsion spreads).

One skilled in the art will recognize that various processes may be usedto hydrogenate carbohydrates. One suitable method includes contactingthe carbohydrate with hydrogen or hydrogen mixed with a suitable gas anda catalyst under conditions sufficient in a hydrogenation reactor toform a hydrogenated product. The hydrogenation catalyst generally caninclude Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or anycombination thereof, either alone or with promoters such as W, Mo, Au,Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof.Other effective hydrogenation catalyst materials include eithersupported nickel or ruthenium modified with rhenium. In an embodiment,the hydrogenation catalyst also includes any one of the supports,depending on the desired functionality of the catalyst. Thehydrogenation catalysts may be prepared by methods known to those ofordinary skill in the art.

In some embodiments the hydrogenation catalyst includes a supportedGroup VIII metal catalyst and a metal sponge material (e.g., a spongenickel catalyst). Raney nickel provides an example of an activatedsponge nickel catalyst suitable for use in this invention. In otherembodiment, the hydrogenation reaction in the invention is performedusing a catalyst comprising a nickel-rhenium catalyst or atungsten-modified nickel catalyst. One example of a suitable catalystfor the hydrogenation reaction of the invention is a carbon-supportednickel-rhenium catalyst.

In an embodiment, a suitable Raney nickel catalyst may be prepared bytreating an alloy of approximately equal amounts by weight of nickel andaluminum with an aqueous alkali solution, e.g., containing about 25weight % of sodium hydroxide. The aluminum is selectively dissolved bythe aqueous alkali solution resulting in a sponge shaped materialcomprising mostly nickel with minor amounts of aluminum. The initialalloy includes promoter metals (i.e., molybdenum or chromium) in theamount such that about 1 to 2 weight % remains in the formed spongenickel catalyst. In another embodiment, the hydrogenation catalyst isprepared using a solution of ruthenium (III) nitrosylnitrate, ruthenium(III) chloride in water to impregnate a suitable support material. Thesolution is then dried to form a solid having a water content of lessthan about 1% by weight. The solid may then be reduced at atmosphericpressure in a hydrogen stream at 300° C. (uncalcined) or 400° C.(calcined) in a rotary ball furnace for 4 hours. After cooling andrendering the catalyst inert with nitrogen, 5% by volume of oxygen innitrogen is passed over the catalyst for 2 hours.

In certain embodiments, the catalyst described includes a catalystsupport. The catalyst support stabilizes and supports the catalyst. Thetype of catalyst support used depends on the chosen catalyst and thereaction conditions. Suitable supports for the invention include, butare not limited to, carbon, silica, silica-alumina, zirconia, titania,ceria, vanadia, nitride, boron nitride, heteropolyacids, hydroxyapatite,zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene andany combination thereof.

The catalysts used in this invention can be prepared using conventionalmethods known to those in the art. Suitable methods may include, but arenot limited to, incipient wetting, evaporative impregnation, chemicalvapor deposition, wash-coating, magnetron sputtering techniques, and thelike.

The conditions for which to carry out the hydrogenation reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate reaction conditions. In general, thehydrogenation reaction is conducted at temperatures of 80° C. to 250°C., and preferably at 90° C. to 200° C., and most preferably at 100° C.to 150° C. In some embodiments, the hydrogenation reaction is conductedat pressures from 500 KPa to 14000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention may include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof. As used herein, theterm “external hydrogen” refers to hydrogen that does not originate fromthe biomass reaction itself, but rather is added to the system fromanother source.

In some embodiments of the invention, it is desirable to convert thestarting carbohydrate to a smaller molecule that will be more readilyconverted to desired higher hydrocarbons. One suitable method for thisconversion is through a hydrogenolysis reaction. Various processes areknown for performing hydrogenolysis of carbohydrates. One suitablemethod includes contacting a carbohydrate with hydrogen or hydrogenmixed with a suitable gas and a hydrogenolysis catalyst in ahydrogenolysis reactor under conditions sufficient to form a reactionproduct comprising smaller molecules or polyols. As used herein, theterm “smaller molecules or polyols” includes any molecule that has asmaller molecular weight, which can include a smaller number of carbonatoms or oxygen atoms than the starting carbohydrate. In an embodiment,the reaction products include smaller molecules that include polyols andalcohols. Someone of ordinary skill in the art would be able to choosethe appropriate method by which to carry out the hydrogenolysisreaction.

In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may beconverted to propylene glycol, ethylene glycol, and glycerol using ahydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo,W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or anycombination thereof, either alone or with promoters such as Au, Ag, Cr,Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. Thehydrogenolysis catalyst may also include a carbonaceous pyropolymercatalyst containing transition metals (e.g., chromium, molybdemum,tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals(e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium,iridium, and osmium). In certain embodiments, the hydrogenolysiscatalyst may include any of the above metals combined with an alkalineearth metal oxide or adhered to a catalytically active support. Incertain embodiments, the catalyst described in the hydrogenolysisreaction may include a catalyst support as described above for thehydrogenation reaction.

The conditions for which to carry out the hydrogenolysis reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate conditions to use to carry out thereaction. In general, they hydrogenolysis reaction is conducted attemperatures of 110° C. to 300° C., and preferably at 170° C. to 220°C., and most preferably at 200° C. to 225° C. In some embodiments, thehydrogenolysis reaction is conducted under basic conditions, preferablyat a pH of 8 to 13, and even more preferably at a pH of 10 to 12. Insome embodiments, the hydrogenolysis reaction is conducted at pressuresin a range between 60 KPa and 16500 KPa, and preferably in a rangebetween 1700 KPa and 14000 KPa, and even more preferably between 4800KPa and 11000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention can include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof.

In some embodiments, the reaction products discussed above may beconverted into higher hydrocarbons through a condensation reaction in acondensation reactor. In such embodiments, condensation of the reactionproducts occurs in the presence of a catalyst capable of forming higherhydrocarbons. While not intending to be limited by theory, it isbelieved that the production of higher hydrocarbons proceeds through astepwise addition reaction including the formation of carbon-carbon, orcarbon-oxygen bond. The resulting reaction products include any numberof compounds containing these moieties, as described in more detailbelow.

In certain embodiments, suitable condensation catalysts include an acidcatalyst, a base catalyst, or an acid/base catalyst. As used herein, theterm “acid/base catalyst” refers to a catalyst that has both an acid anda base functionality. In some embodiments the condensation catalyst caninclude, without limitation, zeolites, carbides, nitrides, zirconia,alumina, silica, aluminosilicates, phosphates, titanium oxides, zincoxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandiumoxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides,hydroxides, heteropolyacids, inorganic acids, acid modified resins, basemodified resins, and any combination thereof. In some embodiments, thecondensation catalyst can also include a modifier. Suitable modifiersinclude La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and anycombination thereof. In some embodiments, the condensation catalyst canalso include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe,Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys,and any combination thereof.

In certain embodiments, the catalyst described in the condensationreaction may include a catalyst support as described above for thehydrogenation reaction. In certain embodiments, the condensationcatalyst is self-supporting. As used herein, the term “self-supporting”means that the catalyst does not need another material to serve assupport. In other embodiments, the condensation catalyst in used inconjunction with a separate support suitable for suspending thecatalyst. In an embodiment, the condensation catalyst support is silica.

The conditions under which the condensation reaction occurs will varybased on the type of starting material and the desired products. One ofordinary skill in the art, with the benefit of this disclosure, willrecognize the appropriate conditions to use to carry out the reaction.In some embodiments, the condensation reaction is carried out at atemperature at which thermodynamics for the proposed reaction arefavorable. The temperature for the condensation reaction will varydepending on the specific starting polyol or alcohol. In someembodiments, the temperature for the condensation reaction is in a rangefrom 80° C. to 500° C., and preferably from 125° C. to 450° C., and mostpreferably from 125° C. to 250° C. In some embodiments, the condensationreaction is conducted at pressures in a range between 0 Kpa to 9000 KPa,and preferably in a range between 0 KPa and 7000 KPa, and even morepreferably between 0 KPa and 5000 KPa.

The higher alkanes formed by the invention include, but are not limitedto, branched or straight chain alkanes that have from 4 to 30 carbonatoms, branched or straight chain alkenes that have from 4 to 30 carbonatoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenesthat have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, andketones. Suitable alkanes include, but are not limited to, butane,pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane,3-methylpentane, 2,2,-dimethylbutane, 2,3-dimethylbutane, heptane,heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane,2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane,decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene,tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane,nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane,doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, andisomers thereof. Some of these products may be suitable for use asfuels.

In some embodiments, the cycloalkanes and the cycloalkenes areunsubstituted. In other embodiments, the cycloalkanes and cycloalkenesare mono-substituted. In still other embodiments, the cycloalkanes andcycloalkenes are multi-substituted. In the embodiments comprising thesubstituted cycloalkanes and cycloalkenes, the substituted groupincludes, without limitation, a branched or straight chain alkyl having1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to12 carbon atoms, a phenyl, and any combination thereof. Suitablecycloalkanes and cycloalkenes include, but are not limited to,cyclopentane, cyclopentene, cyclohexane, cyclohexene,methyl-cyclopentane, methyl-cyclopentene, ethyl-cyclopentane,ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers andany combination thereof.

In some embodiments, the aryls formed are unsubstituted. In anotherembodiment, the aryls formed are mono-substituted. In the embodimentscomprising the substituted aryls, the substituted group includes,without limitation, a branched or straight chain alkyl having 1 to 12carbon atoms, a branched or straight chain alkylene having 1 to 12carbon atoms, a phenyl, and any combination thereof. Suitable aryls forthe invention include, but are not limited to, benzene, toluene, xylene,ethyl benzene, para xylene, meta xylene, and any combination thereof.

The alcohols produced in the invention have from 4 to 30 carbon atoms.In some embodiments, the alcohols are cyclic. In other embodiments, thealcohols are branched. In another embodiment, the alcohols are straightchained. Suitable alcohols for the invention include, but are notlimited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol,decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol,hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol,uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomersthereof.

The ketones produced in the invention have from 4 to 30 carbon atoms. Inan embodiment, the ketones are cyclic. In another embodiment, theketones are branched. In another embodiment, the ketones are straightchained. Suitable ketones for the invention include, but are not limitedto, butanone, pentanone, hexanone, heptanone, octanone, nonanone,decanone, undecanone, dodecanone, tridecanone, tetradecanone,pentadecanone, hexadecanone, heptyldecanone, octyldecanone,nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone,tetraeicosanone, and isomers thereof.

Another such chemical modification is interesterification. Naturallyproduced glycerolipids do not have a uniform distribution of fatty acidconstituents. In the context of oils, interesterification refers to theexchange of acyl radicals between two esters of different glycerolipids.The interesterification process provides a mechanism by which the fattyacid constituents of a mixture of glycerolipids can be rearranged tomodify the distribution pattern. Interesterification is a well-knownchemical process, and generally comprises heating (to about 200° C.) amixture of oils for a period (e.g, 30 minutes) in the presence of acatalyst, such as an alkali metal or alkali metal alkylate (e.g., sodiummethoxide). This process can be used to randomize the distributionpattern of the fatty acid constituents of an oil mixture, or can bedirected to produce a desired distribution pattern. This method ofchemical modification of lipids can be performed on materials providedherein, such as microbial biomass with a percentage of dry cell weightas lipid at least 20%.

Directed interesterification, in which a specific distribution patternof fatty acids is sought, can be performed by maintaining the oilmixture at a temperature below the melting point of some TAGs whichmight occur. This results in selective crystallization of these TAGs,which effectively removes them from the reaction mixture as theycrystallize. The process can be continued until most of the fatty acidsin the oil have precipitated, for example. A directedinteresterification process can be used, for example, to produce aproduct with a lower calorie content via the substitution oflonger-chain fatty acids with shorter-chain counterparts. Directedinteresterification can also be used to produce a product with a mixtureof fats that can provide desired melting characteristics and structuralfeatures sought in food additives or products (e.g., margarine) withoutresorting to hydrogenation, which can produce unwanted trans isomers.

Interesterification of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,080,853 (Nondigestible fat substitutes); U.S. Pat. No.4,288,378 (Peanut butter stabilizer); U.S. Pat. No. 5,391,383 (Ediblespray oil); U.S. Pat. No. 6,022,577 (Edible fats for food products);U.S. Pat. No. 5,434,278 (Edible fats for food products); U.S. Pat. No.5,268,192 (Low calorie nut products); U.S. Pat. No. 5,258,197 (Reducecalorie edible compositions); U.S. Pat. No. 4,335,156 (Edible fatproduct); U.S. Pat. No. 7,288,278 (Food additives or medicaments); U.S.Pat. No. 7,115,760 (Fractionation process); U.S. Pat. No. 6,808,737(Structural fats); U.S. Pat. No. 5,888,947 (Engine lubricants); U.S.Pat. No. 5,686,131 (Edible oil mixtures); and U.S. Pat. No. 4,603,188(Curable urethane compositions).

In one embodiment in accordance with the invention, transesterificationof the oil, as described above, is followed by reaction of thetransesterified product with polyol, as reported in U.S. Pat. No.6,465,642, to produce polyol fatty acid polyesters. Such anesterification and separation process may comprise the steps as follows:reacting a lower alkyl ester with polyol in the presence of soap;removing residual soap from the product mixture; water-washing anddrying the product mixture to remove impurities; bleaching the productmixture for refinement; separating at least a portion of the unreactedlower alkyl ester from the polyol fatty acid polyester in the productmixture; and recycling the separated unreacted lower alkyl ester.

Transesterification can also be performed on microbial biomass withshort chain fatty acid esters, as reported in U.S. Pat. No. 6,278,006.In general, transesterification may be performed by adding a short chainfatty acid ester to an oil in the presence of a suitable catalyst andheating the mixture. In some embodiments, the oil comprises about 5% toabout 90% of the reaction mixture by weight. In some embodiments, theshort chain fatty acid esters can be about 10% to about 50% of thereaction mixture by weight. Non-limiting examples of catalysts includebase catalysts, sodium methoxide, acid catalysts including inorganicacids such as sulfuric acid and acidified clays, organic acids such asmethane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid,and acidic resins such as Amberlyst 15. Metals such as sodium andmagnesium, and metal hydrides also are useful catalysts.

Another such chemical modification is hydroxylation, which involves theaddition of water to a double bond resulting in saturation and theincorporation of a hydroxyl moiety. The hydroxylation process provides amechanism for converting one or more fatty acid constituents of aglycerolipid to a hydroxy fatty acid. Hydroxylation can be performed,for example, via the method reported in U.S. Pat. No. 5,576,027.Hydroxylated fatty acids, including castor oil and its derivatives, areuseful as components in several industrial applications, including foodadditives, surfactants, pigment wetting agents, defoaming agents, waterproofing additives, plasticizing agents, cosmetic emulsifying and/ordeodorant agents, as well as in electronics, pharmaceuticals, paints,inks, adhesives, and lubricants. One example of how the hydroxylation ofa glyceride may be performed is as follows: fat may be heated,preferably to about 30-50° C. combined with heptane and maintained attemperature for thirty minutes or more; acetic acid may then be added tothe mixture followed by an aqueous solution of sulfuric acid followed byan aqueous hydrogen peroxide solution which is added in small incrementsto the mixture over one hour; after the aqueous hydrogen peroxide, thetemperature may then be increased to at least about 60° C. and stirredfor at least six hours; after the stirring, the mixture is allowed tosettle and a lower aqueous layer formed by the reaction may be removedwhile the upper heptane layer formed by the reaction may be washed withhot water having a temperature of about 60° C.; the washed heptane layermay then be neutralized with an aqueous potassium hydroxide solution toa pH of about 5 to 7 and then removed by distillation under vacuum; thereaction product may then be dried under vacuum at 100° C. and the driedproduct steam-deodorized under vacuum conditions and filtered at about50° to 60° C. using diatomaceous earth.

Hydroxylation of microbial oils produced by the methods described hereincan be performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. No. 6,590,113 (Oil-based coatings and ink); U.S. Pat. No. 4,049,724(Hydroxylation process); U.S. Pat. No. 6,113,971 (Olive oil butter);U.S. Pat. No. 4,992,189 (Lubricants and lube additives); U.S. Pat. No.5,576,027 (Hydroxylated milk); and U.S. Pat. No. 6,869,597 (Cosmetics).

Hydroxylated glycerolipids can be converted to estolides. Estolidesconsist of a glycerolipid in which a hydroxylated fatty acid constituenthas been esterified to another fatty acid molecule. Conversion ofhydroxylated glycerolipids to estolides can be carried out by warming amixture of glycerolipids and fatty acids and contacting the mixture witha mineral acid, as described by Isbell et al., JAOCS 71(2):169-174(1994). Estolides are useful in a variety of applications, includingwithout limitation those reported in the following: U.S. Pat. No.7,196,124 (Elastomeric materials and floor coverings); U.S. Pat. No.5,458,795 (Thickened oils for high-temperature applications); U.S. Pat.No. 5,451,332 (Fluids for industrial applications); U.S. Pat. No.5,427,704 (Fuel additives); and U.S. Pat. No. 5,380,894 (Lubricants,greases, plasticizers, and printing inks).

Another such chemical modification is olefin metathesis. In olefinmetathesis, a catalyst severs the alkylidene carbons in an alkene(olefin) and forms new alkenes by pairing each of them with differentalkylidine carbons. The olefin metathesis reaction provides a mechanismfor processes such as truncating unsaturated fatty acid alkyl chains atalkenes by ethenolysis, cross-linking fatty acids through alkenelinkages by self-metathesis, and incorporating new functional groups onfatty acids by cross-metathesis with derivatized alkenes.

In conjunction with other reactions, such as transesterification andhydrogenation, olefin metathesis can transform unsaturated glycerolipidsinto diverse end products. These products include glycerolipid oligomersfor waxes; short-chain glycerolipids for lubricants; homo- andhetero-bifunctional alkyl chains for chemicals and polymers; short-chainesters for biofuel; and short-chain hydrocarbons for jet fuel. Olefinmetathesis can be performed on triacylglycerols and fatty acidderivatives, for example, using the catalysts and methods reported inU.S. Pat. No. 7,119,216, US Patent Pub. No. 2010/0160506, and U.S.Patent Pub. No. 2010/0145086.

Olefin metathesis of bio-oils generally comprises adding a solution ofRu catalyst at a loading of about 10 to 250 ppm under inert conditionsto unsaturated fatty acid esters in the presence (cross-metathesis) orabsence (self-metathesis) of other alkenes. The reactions are typicallyallowed to proceed from hours to days and ultimately yield adistribution of alkene products. One example of how olefin metathesismay be performed on a fatty acid derivative is as follows: A solution ofthe first generation Grubbs Catalyst(dichloro[2(1-methylethoxy-α-O)phenyl]methylene-α-C](tricyclohexyl-phosphine) in toluene at a catalyst loading of 222 ppmmay be added to a vessel containing degassed and dried methyl oleate.Then the vessel may be pressurized with about 60 psig of ethylene gasand maintained at or below about 30° C. for 3 hours, wherebyapproximately a 50% yield of methyl 9-decenoate may be produced.

Olefin metathesis of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: PatentApp. PCT/US07/081427 (α-olefin fatty acids) and U.S. patent applicationSer. No. 12/281,938 (petroleum creams), Ser. No. 12/281,931 (paintballgun capsules), Ser. No. 12/653,742 (plasticizers and lubricants), Ser.No. 12/422,096 (bifunctional organic compounds), and Ser. No. 11/795,052(candle wax).

Other chemical reactions that can be performed on microbial oils includereacting triacylglycerols with a cyclopropanating agent to enhancefluidity and/or oxidative stability, as reported in U.S. Pat. No.6,051,539; manufacturing of waxes from triacylglycerols, as reported inU.S. Pat. No. 6,770,104; and epoxidation of triacylglycerols, asreported in “The effect of fatty acid composition on the acrylationkinetics of epoxidized triacylglycerols”, Journal of the American OilChemists' Society, 79:1, 59-63, (2001) and Free Radical Biology andMedicine, 37:1, 104-114 (2004).

The generation of oil-bearing microbial biomass for fuel and chemicalproducts as described above results in the production of delipidatedbiomass meal. Delipidated meal is a byproduct of preparing algal oil andis useful as animal feed for farm animals, e.g., ruminants, poultry,swine and aquaculture. The resulting meal, although of reduced oilcontent, still contains high quality proteins, carbohydrates, fiber,ash, residual oil and other nutrients appropriate for an animal feed.Because the cells are predominantly lysed by the oil separation process,the delipidated meal is easily digestible by such animals. Delipidatedmeal can optionally be combined with other ingredients, such as grain,in an animal feed. Because delipidated meal has a powdery consistency,it can be pressed into pellets using an extruder or expander or anothertype of machine, which are commercially available.

The triglyceride oils provided herein also find use in the preparationof fexible foam polyurethanes.

In one embodiment, provided is a method for preparing a polyurethanefoam, the method comprising:

a) providing a microalgal oil comprising triacylglycerides having afatty acid profile of at least 80% 18:1 fatty acids and less than 4%18:2 fatty acids, the triglycerides further having an iodine value of 75or less and/or is non-hydrogenated;

b) subjecting the triacylglycerides to an epoxidation reaction to formepoxides;

c) subjecting the epoxides to a ring opening reaction to form microalgalderived polyols, wherein the microalgal derived polyols are liquids atroom temperature;

d) blending the microalgal derived polyols with petroleum derivedpolyols to form blended polyols; and

e) subjecting the blended polyols to an isocyanate condensation reactionto form the polyurethane foam.

In some embodiments, the polyurethane foam has less than 25% loss inthickness after being compressed to 75% thickness at 70° C. and 5%relative humidity for 22 hours in accordance with American Society forTesting and Materials standard ASTM D-3574. In some embodiments, theloss in thickness is less than a 20%. In other embodiments, the loss inthickness is less than a 15%.

In some embodiments, the polyurethane foam is resistant to yellowing. Insome embodiments, the polyurethane foam maintains a yellowness index of31 or less after being exposed to ambient light for 18 weeks inaccordance with American Society for Testing and Materials standard ASTMD-E313.

In some embodiments, the triacylglycerides have an iodine value of70-75. In some embodiments, the triacylglycerides have an iodine valueof 74-75. The iodine value provides a measure of the degree ofunsaturation, and is the amount of grams of iodine that will react with100 grams of sample.

In some embodiments, the polyurethane foam is prepared from thetriacylglycerides having a fatty acid profile of have less than 1%, lessthan 0.1%, or less than 0.01% trans fatty acids or wherein thetriacylglyceride is non-hydrogenated.

In some embodiments, the triacylglycerides have a fatty acid profile ofless than 3%, 2%, less than 1%, less than 0.1%, less than 0.01%, or lessthan 0.001% 18:2 fatty acids. In some embodiments, the polyurethane foamis prepared from the triacylglycerides having a fatty acid profile ofhave less than 0.01% trans fatty acids and less than 0.1% or less than0.01% 18:2 fatty acids.

In some embodiments, the triacylglycerides have a fatty acid profile ofless than 15%, less than 14%, less than 13%, less than 12%, less than11%, or less than 10% saturated fatty acids.

In some embodiments, the triacylglycerides have a fatty acid profile ofless than 5%, less than 4%, less than 3%, or less than 2% 18:0 fattyacids.

In some embodiments, the polyurethane foam is prepared from thetriacylglycerides having a fatty acid profile of at least 85% 18:1 fattyacids.

In some embodiments, the triacylglycerides having a fatty acid profileof at least 85% 18:1 fatty acids and 1% or less 18:2 fatty acids.

In some embodiments, the triacylglycerides having a fatty acid profileof at least 85% 18:1 fatty acids and 0.1% or less 18:2 fatty acids.

In some embodiments, microalgal oil is obtained from microalgaecultivated under heterotrophic conditions. In some embodiments themicroalgal oil comprises C29 and C28 sterols, wherein the amount of C28sterols is greater than C29 sterols. In some embodiments the microalgalis Parachlorella, Prototheca, or Chlorella. In some embodiments themicroalgae is Prototheca. In some embodiments the microalgae isPrototheca moriformis. In some embodiments, the microalgal oil isobtained from a microalgae comprising recombinant nucleic acids operableto reduce the activity a Δ12 fatty acid desaturase. In other embodimentsthe microalgae further comprises recombinant nucleic acids operable toreduce the activity a FATA thioesterase.

In some embodiments, the polyurethane foam is prepared by subjectingtriacylglycerides provided herein to an epoxidation reaction to formepoxides; subjecting the the epoxides to an epoxide ring openingreaction to form polyol intermediates that are substantially free fromsolids at room temperature; and subjecting the polyol intermediates to acondensation reaction with an isocyanate to form polyurethanes. In someembodiments, the epoxide ring opening reaction comprises treatment ofthe epoxides with methanol.

In the epoxidation reactions the triacylglycerides are exposed to anoxidant such as a peroxyacid or hydroperoxide. Examples of peroxyacidthat may be used include peroxyformic acid, peroxyacetic acid,trifluoroperoxyacetic acid, benzyloxyperoxyformic acid,3,5-dinitroperoxybenzoic acid, m-chloroperoxybenzoic acid, andcombinations thereof. The peroxy acid may be added directly to thereaction, or may be formed in-situ by reacting a hydroperoxide compoundwith an acid such as formic acid, benzoic acid, acetic acid or fattyacids such as oleic acid. Examples of typical hydroperoxides that may beutilized include hydrogen peroxide, tert-butylhydroperoxide,triphenysilylhydroperoxide, cumylhydroperoxide, and combinationsthereof. Most preferably hydrogen peroxide will be used. Preferably, theamount of acid used to form the peroxyacid is from about 0.25 to about1.0 moles of acid per mole of carbon-carbon double bonds in the oil, andmore preferably from about 0.45 to about 0.55 moles of acid per mole ofcarbon-carbon double bonds in the oil. Preferably, the amount ofhydroperoxide used to form the peroxy acid is 0.5 to 1.5 moles ofhydroperoxide per mole of double bonds in the oil, and more preferably0.8 to 1.2 moles of hydroperoxide per mole of double bonds in the oil.

The fully-epoxidized oil is reacted with a ring opener under suitableconditions to provide a polyol. A ring-opening catalyst is typicallyutilized. The ring-opening catalyst preferably is an acid catalyst.Representative examples of ring-opening acid catalysts include Lewisacids and Bronsted acids. Examples of Bronsted acids includehydrofluoroboric acid (HBF₄), trifluoroacetic acid, sulfuric acid,hydrochloric acid phosphoric acid, phosphorous acid, hypophosphorousacid, boronic acids, sulfonic acids (for example, para-toluene sulfonicacid, methanesulfonic acid, and trifluoromethane sulfonic acid), andcarboxylic acids (for example, formic acid and acetic acid). Otherexamples of acids include fluoroboric acid (tetrafluoroboric acid,H₃OBF₄) and copper tetrafluoroborate. Examples of Lewis acids includephosphorous trichloride and boron halides (for example, borontrifluoride). Ion exchange resins in the protic form may also be used.The ring-opening catalyst is typically present in an amount ranging from0.01 wt % to 0.3 wt %, preferably from about 0.05 wt % to 0.15 wt %,based on the total weight of the reaction mixture.

The ring-opener may include alcohols, water, and other compounds havingone or more nucleophilic groups. Combinations of ring-openers may beused. Exemplary ring openers include C1-C4 monohydric alcohols,monoalkyl ethers of ethylene glycol (e.g. methyl cellosolve, butylcellosolve, and the like). Preferably, monohydric alcohols havingbetween one and six carbon atoms are utilized, more preferably one tofour carbon atoms, further more preferably methanol, ethanol, orpropanol, most preferably methanol. Also, mono- and di-carboxylic acidsmay be used. For example, mono-carboxylic acids, such as formic acid,acetic acid, proprionic acid, and butyric acid. Also, polyols may beutilized as the ring opener. Polyols having two or less hydroxyl groupsper molecule are preferred. Examples of preferable polyols includeethylene glycol, propylene glycol, 1,3 propanediol, buylene-glycol,1,4-butane diol, 1,5-pentanediol, 1,6-hexanediol, polyethylene glycoland polypropylene glycol, and vegetable oil-based polyols (for example,polyols as described in U.S. Pat. No. 6,433,121). Also, during the ringopening reaction the polyols created during the ring opening reactionwill react with each other to polymerize and form oligomers.

In some embodiments the microalgal derived polyols are non-hazy liquidssubstantially free from solids. In some embodiments the microalgalderived polyols comprise at least 20% by weight of the blended polyols.In some embodiments the microalgal derived polyols comprise 20 to 50% byweight of the blended polyols.

In some embodiments, the petroleum derived polyols are polyetherpolyols. Petroleum-derived polyols include polyether polyols andpolyester polyols. Polyether polyols and polyester polyols are known toone of skill in the art. Polyether polyols are preferably utilized.Examples of polyether polyols include polyols sold under the marksJEFFOL (available from Huntsman), VORANOL (available from The DowChemical Company), PLURACOL and PLURACEL (available from BASF), ARCOL,HYPERLITE, MUTRANOL, ULTRACEL, SOFTCELL, and ACCLAIM (available from theBayer Corporation), and CARADOL (available from Shell Chemicals).

In some embodiments, the polyols are mixed with a catalyst prior to theisocyanate condensation reaction. Exemplary catalysts include tertiaryamine compounds and organometallic compounds. Specific examples ofuseful tertiary amine compounds include triethylenediamine,N-methylmorpholine, N-ethylmorpholine, diethyl ethanolamine, N-cocomorpholine, 1-methyl-4-dimethylaminoethyl piperazine,3-methoxy-N-dimethylpropylamine, N,N-diethyl-3diethylaminopropylamine,dimethylbenzyl amine, bis(2-dimethylaminoethyl)ether, and the like.Tertiary amine catalysts are advantageously used in an amount from 0.01to 5 parts, preferably from 0.05 to 2 parts per hundred (100) parts byweight of the active hydrogen-containing composition in the formulation.Specific examples of useful organometallic catalysts include organicsalts of metals such as tin, bismuth, iron, zinc, and the like.Organotin catalysts are preferred. Examples of organotin catalystsinclude dimethyltindilaurate, dibutyltindilaurate, and stannous octoate.Other preferred catalyst include those disclosed in U.S. Pat. No.2,846,408, which is hereby incorporated by reference for its teachingsregarding organometallic catalysts useful for polyurethane reactions.Preferably, 0.001 to 1.0 parts by weight of an organometallic catalystis used per hundred (100) parts by weight of the activehydrogen-containing composition in the formulation. In some embodiments,the catalyst is an amine catalyst. In some embodiments, the aminecatalyst is a diethanolamine catalyst. In some embodiments, the moleratio of the amine catalyst to the microalgal polyol is greater than3:1.

The polyurethanes provided herein can also be prepared with and/orcontain additives, alcohols, blowing agents, and surfactants. Additivesinclude release agents, anti-oxidants, flame retardant additives, cellsize control agents, cell opening agents, colorants, preservatives,static dissipative agents, plasticizers, and crosslinking agents.Blowing agents include low temperature boiling point solvents (e.g. lowmolecular weight organic compounds such as halogenated alkanes, acetone,and isopentane) for generating gases to expand the polyurethane foamduring its synthesis. In some embodiments the surfactant is a siliconsurfactant.

Methods and components for forming polyurethanes can also be found in US2005/0282921.

In some embodiments, the polyurethanes provided herein contain fillersto increase foam density. Suitable fillers include inorganic materialssuch as barium sulfate, sand, or clay.

In some embodiments, the polyurethane foams provided herein are flexibleslabstock foam.

In some embodiments, provided is an article comprising a polyurethanefoam provided herein. In some embodiments the article is a support orsafety application selected from seat cushion, seat backing, headrest,armrest, automotive headliner, automotive soft instrument panel,mattress, pillow, carpet backing, packaging, sports equipment, andhelmet liner.

In some embodiments, the polyurethane foams provided herein find use asseat cushions and in automotive applications.

The invention, having been described in detail above, is exemplified inthe following examples, which are offered to illustrate, but not tolimit, the claimed invention.

XIV. Examples Example 1 Fatty Acid Analysis by Fatty Acid Methyl EsterDetection

Lipid samples were prepared from dried biomass. 20-40 mg of driedbiomass was resuspended in 2 mL of 5% H₂SO₄ in MeOH, and 200 ul oftoluene containing an appropriate amount of a suitable internal standard(C19:0) was added. The mixture was sonicated briefly to disperse thebiomass, then heated at 70-75° C. for 3.5 hours. 2 mL of heptane wasadded to extract the fatty acid methyl esters, followed by addition of 2mL of 6% K₂CO₃ (aq) to neutralize the acid. The mixture was agitatedvigorously, and a portion of the upper layer was transferred to a vialcontaining Na₂SO₄ (anhydrous) for gas chromatography analysis usingstandard FAME GC/FID (fatty acid methyl ester gas chromatography flameionization detection) methods. Fatty acid profiles reported below weredetermined by this method.

Example 2 Triacylglyceride Purification from Oil and Methods forTriacylglyceride Lipase Digestion

The triacylglyceride (TAG) fraction of each oil sample was isolated bydissolving ˜10 mg of oil in dichloromethane and loading it onto aBond-Elut aminopropyl solid-phase extraction cartridge (500 mg)preconditioned with heptane. TAGs were eluted with dicholoromethane-MeOH(1:1) into a collection tube, while polar lipids were retained on thecolumn. The solvent was removed with a stream of nitrogen gas. Trisbuffer and 2 mg porcine pancreatic lipase (Type II, Sigma, 100-400units/mg) were added to the TAG fraction, followed by addition of bilesalt and calcium chloride solutions. The porcine pancreatic lipasecleaves sn-1 and sn-3 fatty acids, thereby generating2-monoacylglycerides and free fatty acids. This mixture was heated withagitation at 40° C. for three minutes, cooled briefly, then quenchedwith 6 N HCl. The mixture was then extracted with diethyl ether and theether layer was washed with water then dried over sodium sulfate. Thesolvent was removed with a stream of nitrogen. To isolate themonoacylglyceride (MAG) fraction, the residue was dissolved in heptaneand loaded onto a second aminopropyl solid phase extraction cartridgepretreated with heptane. Residual TAGs were eluted with diethylether-dichloromethane-heptane (1:9:40), diacylglycerides (DAGs) wereeluted with ethyl acetate-heptane (1:4), and MAGs were eluted from thecartridge with dichloromethane-methanol (2:1). The resulting MAG, DAG,and TAG fractions were then concentrated to dryness with a stream ofnitrogen and subjected to routine direct transesterification method ofGC/FID analysis as described in Example 1.

Example 3 Engineering Microorganisms for Fatty Acid and SN-2 ProfilesIncreased in Lauric Acid Through Exogenous LPAAT Expression

This example describes the use of recombinant polynucleotides thatencode a C. nucifera 1-acyl-sn-glycerol-3-phosphate acyltransferase (CnLPAAT) enzyme to engineer a microorganism in which the fatty acidprofile and the sn-2 profile of the transformed microorganism has beenenriched in lauric acid.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain A, was initially transformed with the plasmid construct pSZ1283according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1283, described inPCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696 herebyincorporated by reference, comprised the coding sequence of the Cupheawrightii FATB2 (CwTE2) thioesterase (SEQ ID NO: 10), 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe nuclear genome, and a S. cerevisiae suc2 sucrose invertase codingregion (SEQ ID NO: 4), to express the protein sequence given in SEQ IDNO: 3, under the control of C. reinhardtii β-tubulin promoter/5′UTR (SEQID NO: 5) and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO:6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO:7 and served as a selectable marker. The CwTE2 protein coding sequenceto express the protein sequence given in SEQ ID NO: 11, was under thecontrol of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C.vulgaris nitrate reductase 3′UTR. The protein coding regions of CwTE2and suc2 were codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

Upon transformation of pSZ1283 into Strain A, positive clones wereselected on agar plates with sucrose as the sole carbon source. Primarytransformants were then clonally purified and a single transformant,Strain B, was selected for further genetic modification. Thisgenetically engineered strain was transformed with plasmid constructpSZ2046 to interrupt the pLoop genomic locus of Strain B. ConstructpSZ2046 comprised the coding sequence of the C. nucifera1-acyl-sn-glycerol-3-phosphate acyltransferase (Cn LPAAT) enzyme (SEQ IDNO: 12), 5′ (SEQ ID NO: 13) and 3′ (SEQ ID NO: 14) homologousrecombination targeting sequences (flanking the construct) to the pLoopgenomic region for integration into the nuclear genome, and a neomycinresistance protein-coding sequence under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 5), and Chlorella vulgaris nitratereductase 3′ UTR (SEQ ID NO: 6). This NeoR expression cassette is listedas SEQ ID NO: 15 and served as a selectable marker. The Cn LPAAT proteincoding sequence was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′UTR.The protein coding regions of Cn LPAAT and NeoR were codon optimized toreflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genesas described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. The amino acid sequence of CnLPAAT is provided as SEQ ID NO: 16.

Upon transformation of pSZ2046 into Strain B, thereby generating StrainC, positive clones were selected on agar plates comprising G418(Geneticin). Individual transformants were clonally purified and grownat pH 7.0 under conditions suitable for lipid production as detailed inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 (U1) grown on glucose as asole carbon source, untransformed Strain B and five pSZ2046 positivetransformants (Strain C, 1-5) are presented in Table 6.

TABLE 6 Effect of LPAAT expression on fatty acid profiles of transformedPrototheca moriformis (UTEX 1435) comprising a mid-chain preferringthioesterase. Area % Strain C- Strain C- Strain C- Strain C- Strain C-Fatty acid U1 Strain B 1 2 3 4 5 C10:0 0.01 5.53 11.37 11.47 10.84 11.1311.12 C12:0 0.04 31.04 46.63 46.47 45.84 45.80 45.67 C14:0 1.27 15.9915.14 15.12 15.20 15.19 15.07 C16:0 27.20 12.49 7.05 7.03 7.30 7.20 7.19C18:0 3.85 1.30 0.71 0.72 0.74 0.74 0.74 C18:1 58.70 24.39 10.26 10.4110.95 11.31 11.45 C18:2 7.18 7.79 7.05 6.93 7.30 6.88 7.01 C10-C12 0.5036.57 58.00 57.94 56.68 56.93 56.79

As shown in Table 6, the fatty acid profile of Strain B expressing CwTE2showed increased composition of C10:0, C12:0, and C14:0 fatty acids anda decrease in C16:0, C18:0, and C18:1 fatty acids relative to the fattyacid profile of the untransformed UTEX 1435 strain. The impact ofadditional genetic modification on the fatty acid profile of thetransformed strains, namely the expression of CnLPAAT in Strain B, is astill further increase in the composition of C10:0 and C12:0 fattyacids, a still further decrease in C16:0, C18:0, and C18:1 fatty acids,but no significant effect on the C14:0 fatty acid composition. Thesedata indicate that the CnLPAAT shows substrate preference in the contextof a microbial host organism.

The untransformed P. moriformis Strain A is characterized by a fattyacid profile comprising less than 0.5% C12 fatty acids and less than 1%C10-C12 fatty acids. In contrast, the fatty acid profile of Strain Bexpressing a C. wrightii thioesterase comprised 31% C12:0 fatty acids,with C10-C12 fatty acids comprising greater than 36% of the total fattyacids. Further, fatty acid profiles of Strain C, expressing a higherplant thioesterase and a CnLPAAT enzyme, comprised between 45.67% and46.63% C12:0 fatty acids, with C10-C12% fatty acids comprising between71 and 73% of total fatty acids. The result of expressing an exogenousthioesterase was a 62-fold increase in the percentage of C12 fatty acidpresent in the engineered microbe. The result of expressing an exogenousthioesterase and exogenous LPAAT was a 92-fold increase in thepercentage of C12 fatty acids present in the engineered microbe.

The TAG fraction of oil samples extracted from Strains A, B, and C wereanalyzed for the sn-2 profile of their triacylglycerides. The TAGs wereextracted and processed as described in Example 2 and analyzed as inExamples 1 and 2. The fatty acid composition and the sn-2 profiles ofthe TAG fraction of oil extracted from Strains A, B, and C (expressed asArea % of total fatty acids) are presented in Table 7. Values notreported are indicated as “n.r.”

TABLE 7 Effect of LPAAT expression on the fatty acid composition and thesn-2 profile of TAGs produced from transformed Prototheca moriformis(UTEX 1435) comprising a mid-chain preferring thioesterase. Strain AreaStrain C % Strain A Strain B (pSZ1500 + fatty (untransformed) (pSZ1500)pSZ2046) acid FA sn-2 profile FA sn-2 profile FA sn-2 profile C10:0 n.r.n.r. 11.9 14.2 12.4 7.1 C12:0 n.r. n.r. 42.4 25 47.9 52.8 C14:0 1.0 0.612 10.4 13.9 9.1 C16:0 23.9 1.6 7.2 1.3 6.1 0.9 C18:0 3.7 0.3 n.r n.r.0.8 0.3 C18:1 64.3 90.5 18.3 36.6 9.9 17.5 C18:2 4.5 5.8 5.8 10.8 6.5 10C18:3 n.r. n.r. n.r. n.r. 1.1 1.6

As shown in Table 7, the fatty acid composition of triglycerides (TAGs)isolated from Strain B expressing CwTE2 was increased for C10:0, C12:0,and C14:0 fatty acids and decrease in C16:0 and C18:1 fatty acidsrelative to the fatty acid profile of TAGs isolated from untransformedStrain A. The impact of additional genetic modification on the fattyacid profile of the transformed strains, namely the expression ofCnLPAAT, was a still further increase in the composition of C10:0 andC12:0 fatty acids, a still further decrease in C16:0, C18:0, and C18:1fatty acids, but no significant effect on the C14:0 fatty acidcomposition. These data indicate that expression of the exogenousCnLPAAT improves the midchain fatty acid profile of transformedmicrobes.

The untransformed P. moriformis Strain A is characterized by an sn-2profile of about 0.6% C14, about 1.6% C16:0, about 0.3% C18:0, about 90%C18:1, and about 5.8% C18:2. In contrast to Strain A, Strain B,expressing a C. wrightii thioesterase is characterized by an sn-2profile that is higher in midchain fatty acids and lower in long chainfatty acids. C12 fatty acids comprised 25% of the sn-2 profile of StrainB. The impact of additional genetic modification on the sn-2 profile ofthe transformed strains, namely the expression of CnLPAAT, was still afurther increase in C12 fatty acids (from 25% to 52.8%), a decrease inC18:1 fatty acids (from 36.6% to 17.5%), and a decrease in C10:0 fattyacids. (The sn-2 profile composition of C14:0 and C16:0 fatty acids wasrelatively similar for Strains B and C.)

These data demonstrate the utility and effectiveness of polynucleotidespermitting exogenous LPAAT expression to alter the fatty acid profile ofengineered microorganisms, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids in microbial cells. Thesedata further demonstrate the utility and effectiveness ofpolynucleotides permitting exogenous thioesterase and exogenous LPAATexpression to alter the sn-2 profile of TAGs produced by microbialcells, in particular in increasing the C12 composition of sn-2 profilesand decreasing the C18:1 composition of sn-2 profiles.

Example 4 Thermal Behavior of Oils Produced from Recombinant Microalgae

FIGS. 1-14 include fatty acid profiles and melting curves of refined,bleached and deodorized oils from genetically engineered Protothecamoriformis strains. In some cases, modifications of the melting curvesare obtained via genetic engineering. For example, some of the oilsproduced have shallower or sharper melting transitions relative tocontrol microalgal oils (i.e., those produced from strains lacking agiven genetic modification) or relative to widely available plant oils.In addition, FIG. 12 shows scanning calorimetry for a high palmitic oilwhen tempered by holding at room temperature for several days (lowertrace) and for the same oil after performing the first scan (uppertrace). The scans ranged from −60° C. to +50° C. with a heating rate of10° C./minute. The differences between the two traces suggests thattempering of the oil caused a change in crystal structure within theoil.

Also of note, FIGS. 10 and 11 show stability testing of RBD-5 and RBD 6.Remarkably, RBD-6, an oil with less than 0.1% 18:2 and 18:3 fatty acidswas substantially stable as measured by the oxidative stability index(AOCS Method Cd 12b-92) even after 36 hours of heating at 110° C.

Table 8, below, gives details of the genetic engineering of the strainsidentified in FIGS. 1-13.

TABLE 8 Genetically engineered strains. RB Z Ulmus Americanathioesterase RBD-1 Cuphea wrightii FATB2 thioesterase driven by amt03RBD-2 Ulmus americana thioesterase RBD-3 Native C. hookerianaC16:0-specific thioesterase with amt03 promoter RBD Y Ulmus Americanathioesterase with Btub promoter RBD X SAD2B knockout with native Cwrightii FAT2B thioesterase, amt03 promoter RBD W SAD2B KO with NativeC. wrightii FATB2 driven by amt03 at insertion site RBD-4 control strainRBD-5 FATA-1 knockout with Carthamus oleate sp. TE driven by amt03promoter at insertion site RBD-6 FADc knockout with Carthamus tinctoriusoleoyl thioesterase

Example 5 Characteristics of Processed Oil Produced from EngineeredMicroorganisms

Methods and effects of transforming Prototheca moriformis (UTEX 1435)with transformation vector pSZ1500 (SEQ ID NO: 17) have been previouslydescribed in PCT Application Nos. PCT/US2011/038463, PCT/US2011/038464,and PCT/US2012/023696.

A classically mutagenized (for higher oil production) derivative ofPrototheca moriformis (UTEX 1435), Strain A, was transformed withpSZ1500 according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1500 comprised nucleotidesequence of the Carthamus tinctorius oleyl-thioesterase (CtOTE) gene,codon-optimized for expression in P. moriformis UTEX 1435. The pSZ1500expression construct included 5′ (SEQ ID NO: 18) and 3′ (SEQ ID NO: 19)homologous recombination targeting sequences (flanking the construct) tothe FADc genomic region for integration into the nuclear genome and a S.cerevisiae suc2 sucrose invertase coding region under the control of C.reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselection marker. The CtOTE coding region was under the control of theP. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgarisnitrate reductase 3′UTR, and the native transit peptide was replacedwith the C. protothecoides stearoyl-ACP desaturase transit peptide (SEQID NO: 9). The protein coding regions of CtOTE and suc2 were codonoptimized to reflect the codon bias inherent in P. moriformis UTEX 1435nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ1500 transformants of Strain A were selected on agar platescontaining sucrose as a sole carbon source, clonally purified, and asingle engineered line, Strain D was selected for analysis. Strain D wasgrown as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Hexaneextraction of the oil from the generated biomass was then performedusing standard methods, and the resulting triglyceride oil wasdetermined to be free of residual hexane. Other methods of extraction ofoil from microalgae using an expeller press are described in PCTApplication No. PCT/US2010/031108 and are hereby incorporated byreference.

Different lots of oil extracted from biomass of Strain D were refined,bleached, and deodorized using standard vegetable oil processingmethods. These procedures generated oil samples RBD437, RBD469, RBD501,RBD 502, RBD503, and RBD529, which were subjected to analytical testingprotocols according to methods defined through the American OilChemists' Society, the American Society for Testing and Materials, andthe International Organization for Standardization. The results of theseanalyses are summarized below in Tables 9-14.

TABLE 9 Analytical results for oil sample RBD469. Method Number TestDescription Results Units AOCS Ca 3a-46 Insoluble impurities <0.01 %AOCS Ca 5a-40 Free Fatty Acids (Oleic) 0.02 % AOCS Ca 5a-40 Acid Value0.04 mg KOH/g AOCS CA 9f-57 Neutral oil 98.9 % D97 Cloud Point −15 degC. D97 Pour Point −18 deg C. Karl Fischer Moisture 0.01 % AOCS Cc 13d-55Chlorophyll <0.01 ppm (modified) Iodine Value 78.3 g I₂/100 g AOCS Cd8b-90 Peroxide Value 0.31 meq/kg ISO 6885 p-Anisidine Value 0.65 AOCS Cc18-80 Dropping Melting point 6.2 deg C. (Mettler) AOCS Cd 11d-96Triglycerides 98.6 % AOCS Cd 11d-96 Monoglyceride <0.01 % AOCS Cd 11d-96Diglycerides 0.68 % AOCS Cd 20-91 Total Polar Compounds 2.62 % IUPAC,2.507 and Oxidized & Polymerized 17.62 % 2.508 Triacylglycerides AOCS Cc9b-55 Flash Point 244 deg C. AOCS Cc 9a-48 Smoke Point 232 deg C. AOCSCd 12b-92 Oxidative Stability Index 31.6 hours Rancimat (110° C.) AOCSCa 6a-40 Unsaponified Matter 2.28 %

RBD469 oil was analyzed for trace element content, solid fat content,and Lovibond color according to AOCS methods. Results of these analysesare presented below in Table 10, Table 10, and Table 11.

TABLE 10 ICP Elemental Analysis of RBD469 oil. Results in Method NumberTest Description ppm AOCS Ca 20-99 and Phosphorus 1.09 AOCS Ca 17-01Calcium 0.1 (modified) Magnesium 0.04 Iron <0.02 Sulfur 28.8 Copper<0.05 Potassium <0.50 Sodium <0.50 Silicon 0.51 Boron 0.06 Aluminum<0.20 Lead <0.20 Lithium <0.02 Nickel <0.20 Vanadium <0.05 Zinc <0.02Arsenic <0.20 Mercury <0.20 Cadmium <0.03 Chromium <0.02 Manganese <0.05Silver <0.05 Titanium <0.05 Selenium <0.50 UOP779 Chloride organic <1UOP779 Chloride inorganic 7.24 AOCS Ba 4e-93 Nitrogen 6.7

TABLE 11 Solid Fat Content of RBD469 Oil Method Number Solid Fat ContentResult AOCS Cd 12b-93 Solid Fat Content 10° C. 0.13% AOCS Cd 12b-93Solid Fat Content 15° C. 0.13% AOCS Cd 12b-93 Solid Fat Content 20° C.0.28% AOCS Cd 12b-93 Solid Fat Content 25° C. 0.14% AOCS Cd 12b-93 SolidFat Content 30° C. 0.08% AOCS Cd 12b-93 Solid Fat Content 35° C. 0.25%

TABLE 12 Lovibond Color of RBD469 Oil Method Number Color Result UnitAOCS Cc 13j-97 red 2 Unit AOCS Cc 13j-97 yellow 27 Unit

RBD469 oil was subjected to transesterification to produce fatty acidmethyl esters (FAMEs). The resulting FAME profile of RBD469 is shown inTable 12.

TABLE 13 FAME Profile of RBD469 Oil Fatty Acid Area % C10 0.01 C12:00.04 C14:0 0.64 C15:0 0.08 C16:0 8.17 C16:1 iso 0.39 C16:1 0.77 C17:00.08 C18:0 1.93 C18:1 85.88 C18:1 iso 0.05 C18:2 0.05 C20:0 0.3 C20:10.06 C20:1 0.44 C22:0 0.11 C23:0 0.03 C24:0 0.1 Total FAMEs Identified99.13

The oil stability indexes (OSI) of 6 RBD oil samples withoutsupplemented antioxidants and 3 RBD oil samples supplemented withantioxidants were analyzed according to the Oil Stability Index AOCSMethod Cd 12b-92. Shown in Table 14 are the results of OSI AOCS Cd12b-92 tests, conducted at 110° C., performed using a Metrohm 873Biodiesel Rancimat. Results, except where indicated with an asterisks(*), are the average of multiple OSI runs. Those samples not analyzedare indicated (NA).

TABLE 14 Oil Stability Index at 110° C. of RBD oil samples with andwithout antioxidants. OSI (hours) for each RBD Sample RBD437 RBD469RBD502 RBD501 RBD503 RBD529 Antioxidant Antioxidant added ConcentrationNone 0 65.41 38.33 72.10 50.32 63.04 26.68 Tocopherol &   35 ppm/ 77.7248.60 82.67 NA NA NA Ascorbyl  16.7 ppm Palmitate Tocopherol &  140 ppm/130.27 81.54* 211.49* NA NA NA Ascorbyl 66.7 ppm Palmitate Tocopherol & 1050 ppm/ >157* >144 242.5* NA NA NA Ascorbyl   500 ppm PalmitateTocopherol   50 ppm NA 46.97 NA NA NA NA TBHQ   20 ppm 63.37 37.4 NA NANA NA

The untransformed P. moriformis (UTEX 1435) acid profile comprises lessthan 60% C18:1 fatty acids and greater than 7% C18:2 fatty acids. Incontrast, Strain D (comprising pSZ1500) exhibited fatty acid profileswith an increased composition of C18:1 fatty acids (to above 85%) and adecrease in C18:2 fatty acids (to less than 0.06%). Upon refining,bleaching, and degumming, RBD oils samples prepared from the oil madefrom strain E exhibited OSI values>26 hrs. With addition ofantioxidants, the OSI of RBD oils prepared from oils of Strain Dincreased from 48.60 hours to greater than 242 hours. In otherexperiments, OSI values of over 400 hours were achieved. Additionalproperties of a low polyunsaturated oil according to embodiments of theinvention are given in FIG. 16.

Example 6 Improving the Levels of Oleic Acid of Engineered MicrobesThrough Allelic Disruption of a Fatty Acid Desaturase and an Acyl-ACPThioesterase

This example describes the use of a transformation vector to disrupt aFATA locus of a Prototheca moriformis strain previously engineered forhigh oleic acid and low linoleic acid production. The transformationcassette used in this example comprised a selectable marker andnucleotide sequences encoding a P. moriformis KASII enzyme to engineermicroorganisms in which the fatty acid profile of the transformedmicroorganism has been altered for further increased oleic acid andlowered palmitic acid levels.

Strain D, described in Example 5 and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 17) according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strainwas used as the host for transformation with construct pSZ2276 toincrease expression of a KASII enzyme while concomitantly ablating anendogenous acyl-ACP thioesterase genetic locus to generate Strain E. ThepSZ2276 transformation construct included 5′ (SEQ ID NO: 20) and 3′ (SEQID NO: 21) homologous recombination targeting sequences (flanking theconstruct) to the FATA1 genomic region for integration into the P.moriformis nuclear genome, an A. thaliana THIC protein coding regionunder the control of the C. protothecoides actin promoter/5′UTR (SEQ IDNO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). ThisAtTHIC expression cassette is listed as SEQ ID NO: 23 and served as aselection marker. The P. moriformis KASII protein coding region wasunder the control of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO:8) and C. vulgaris nitrate reductase 3′UTR, and the native transitpeptide of the KASII enzyme was replaced with the C. protothecoidesstearoyl-ACP desaturase transit peptide (SEQ ID NO: 9). Thecodon-optimized sequence of PmKASII comprising a C. protothecoides 5106stearoyl-ACP desaturase transit peptide is provided the sequencelistings as SEQ ID NO: 24. SEQ ID NO: 25 provides the proteintranslation of SEQ ID NO: 24. The protein coding regions of PmKASII andsuc2 were codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

Primary pSZ2276 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and a single engineered line,strain E was selected for analysis. Strain E was cultivated underheterotrophic lipid production conditions at pH 5.0 and pH 7.0 asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) from the transgenic line arising from transformationwith pSZ2276 into Strain D are shown in Table 15.

TABLE 15 Fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and E engineered for increased oleic acid and loweredlinoleic acid levels. Transfor- mation Area % Fatty Acid StrainConstruct(s) pH C16:0 C18:0 C18:1 C18:2 C20:1 Strain A None pH 5 26.63.3 60.5 6.7 0.07 Strain A None pH 7 28.3 4.1 58 6.5 0.06 Strain DpSZ1500 pH 5 17 3.6 77.1 0.01 0.14 Strain D pSZ1500 pH 7 19.5 5.3 72.60.01 0.09 Strain E pSZ1500 + pH 5 4.1 2.36 88.5 0.04 3.1 pSZ2276 StrainE pSZ1500 + pH 7 2.1 7.8 87.9 0.01 0.5 pSZ2276

As shown in Table 15, targeted interruption of FADc alleles with a CtOTEexpression cassette impacted the fatty acid profiles of transformedmicroorganisms. Fatty acid profiles of Strain D (comprising the pSZ1500transformation vector) showed increased composition of C18:1 fatty acidswith a concomitant decrease in C16:0 and C18:2 fatty acids relative toStrain A. Subsequent transformation of Strain D with pSZ2276 tooverexpress a P. moriformis (UTEX 1435) KASII protein whileconcomitantly ablating a FATA genetic locus (thereby generating StrainE) resulted in still further impact on the fatty acid profiles of thetransformed microorganisms. Fatty acid profiles of Strain E showedincreased composition of C18:1 fatty acids, with a further decrease inC16:0 fatty acids relative to Strains A and D. Propagation of Strain Ein culture conditions at pH 7, to induce expression from the Amt03promoter, resulted in a fatty acid profile that was higher in C18:0 andC18:1 fatty acids and lower in C16:0 fatty acids, relative to the samestrain cultured at pH 5.

These data demonstrate the utility of multiple genetic modifications toimpact the fatty acid profile of a host organism for increased levels ofoleic acid with concomitant decreased levels of linoleic acid andpalmitic acid. Further, this example illustrates the use of recombinantpolynucleotides to target gene interruption of an endogenous FATA allelewith a cassette comprising a pH-regulatable promoter to controlexpression of an exogenous KASII protein-coding region in order to alterthe fatty acid profile of a host microbe.

Example 7 Conditional Expression of a Fatty Acid Desaturase

This example describes the use of a transformation vector toconditionally express a delta 12 fatty acid desaturase (FADs) in aPrototheca moriformis strain previously engineered for high oleic acidand very low linoleic acid production in both seed and lipidproductivity stages of propagation. Very low linoleic acid levels innatural oils are sought for use in certain applications. However,absence of linoleic acid during cell division phase (“seed stage”) of ahost microbe is disadvantageous. Linoleic acid may be supplemented tothe seed medium to hasten cell division and not added during lipidproduction, but this addition imposes unwanted costs. To overcome thischallenge, a transformation cassette was constructed for regulatedexpression of a FAD2 enzyme such that levels of linoleic acidssufficient for cell division could be achieved and oil with very lowlevels of linoleic acids could be produced during the oil productionphase of culture of a microorganism. The transformation cassette used inthis example comprised a selectable marker, a pH-regulatable promoter,and nucleotide sequences encoding a P. moriformis FAD2 enzyme toengineer microorganisms in which the fatty acid profile of thetransformed microorganism has been altered for increased oleic acidproduction and regulatable linoleic acid production.

Strain D, described in Examples 5, 6, and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 17) according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strainwas used as the host for transformation with construct pSZ2413 tointroduce a pH-driven promoter for regulation of a P. moriformis FAD2enzyme. The pSZ2413 transformation construct included 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe P. moriformis nuclear genome, an A. thaliana THIC protein codingregion under the control of the C. protothecoides actin promoter/5′UTR(SEQ ID NO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6).This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served asa selection marker. The P. moriformis FAD2 protein coding region wasunder the control of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO:8) and C. vulgaris nitrate reductase 3′UTR. The codon-optimized sequenceof PmFAD2 is provided the sequence listings as SEQ ID NO: 26. SEQ ID NO:27 provides the protein translation of SEQ ID NO: 26. The protein codingregions of PmFAD2 and suc2 were codon optimized to reflect the codonbias inherent in P. moriformis UTEX 1435 nuclear genes as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ2413 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and isolates of the engineeredline, Strain F were selected for analysis. These isolates werecultivated under heterotrophic lipid production conditions at pH 7.0 (toactivate expression of FAD2 from the PmAmt03 promoter) and at pH 5.0, asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The resulting profile of C18:2 fatty acids(expressed in Area %) from nine representative isolates of transgenicStrain F (F-1 through F-9) arising from transformation with pSZ2413 intoStrain D are shown in Table 16.

TABLE 16 C18:2 fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and F. Area % C18:2 Strain Transformation Construct (s) pH5.0 pH 7.0 A None 6.07 7.26 D pSZ1500 0.01 0.01 F-1 pSZ1500 + pSZ24130.37 5.29 F-2 pSZ1500 + pSZ2413 0.45 6.87 F-3 pSZ1500 + pSZ2413 0.506.79 F-4 pSZ1500 + pSZ2413 0.57 5.06 F-5 pSZ1500 + pSZ2413 0.57 7.58 F-6pSZ1500 + pSZ2413 0.60 6.88 F-7 pSZ1500 + pSZ2413 0.62 6.52 F-8pSZ1500 + pSZ2413 0.63 5.79 F-9 pSZ1500 + pSZ2413 0.77 4.53

As shown in Table 16 the impact of regulated expression of the PmFAD2enzyme, effected though strain culture at different pH levels, is aclear increase in the composition of C18:2 fatty acids in thetransformed microorganism. Linoleic acid comprises about 6% to about7.3% of fatty acids of Strain A. In contrast, Strain D (comprising thepSZ1500 transformation vector to ablate both FAD2 alleles) ischaracterized by a fatty acid profile of 0.01% linoleic acid.Transformation of Strain D with pSZ2413 to generate Strain F results ina recombinant microbe in which the production of linoleic acid isregulated by the Amt03 promoter. Propagation of Strain F isolates inculture conditions at pH 7, to induce FAD2 expression from the Amt03promoter, resulted in a fatty acid profile characterized by about 4.5%to about 7.5% linoleic acid. In contrast, propagation of Strain Fisolates in culture conditions at pH 5 resulted in a fatty acid profilecharacterized by about 0.33 to about 0.77% linoleic acid.

These data demonstrate the utility of and effectiveness of recombinantpolynucleotides permitting conditional expression of a FAD2 enzyme toalter the fatty acid profile of engineered microorganisms, and inparticular in regulating the production of C18:2 fatty acids inmicrobial cells.

Example 8 Analysis of Regiospecific Profile

LC/MS TAG distribution analyses were carried out using a Shimadzu Nexeraultra high performance liquid chromatography system that included aSIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 in-line degasser,and a CTO-20A column oven, coupled to a Shimadzu LCMS 8030 triplequadrupole mass spectrometer equipped with an APCI source. Data wasacquired using a Q3 scan of m/z 350-1050 at a scan speed of 1428 u/secin positive ion mode with the CID gas (argon) pressure set to 230 kPa.The APCI, desolvation line, and heat block temperatures were set to 300,250, and 200° C., respectively, the flow rates of the nebulizing anddrying gases were 3.0 L/min and 5.0 L/min, respectively, and theinterface voltage was 4500 V. Oil samples were dissolved indichloromethane-methanol (1:1) to a concentration of 5 mg/mL, and 0.8 μLof sample was injected onto Shimadzu Shim-pack XR-ODS III (2.2 μm,2.0×200 mm) maintained at 30° C. A linear gradient from 30%dichloromethane-2-propanol (1:1)/acetonitrile to 51%dichloromethane-2-propanol (1:1)/acetonitrile over 27 minutes at 0.48mL/min was used for chromatographic separations.

Example 9 Engineering Microbes for Increased Production of SOS, POP, andPOS Triacylglycerides

This example describes the use of recombinant polynucleotides thatencode a C18:0-preferring Brassica napus thioesterase (BnOTE) enzyme toengineer a microorganism in which the triacylglyceride distribution ofthe transformed microorganism has been enriched in SOS, POS, and POPtriacylglycerides.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain A, was initially transformed with the plasmid construct pSZ1358according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1358, described inPCT/US2012/023696, hereby incorporated by reference, comprised thecoding sequence of the Brassica napus thioesterase (BnOTE) thioesterase(SEQ ID NO: 28), 5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2) homologousrecombination targeting sequences (flanking the construct) to the 6Sgenomic region for integration into the nuclear genome, and a S.cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), toexpress the protein sequence given in SEQ ID NO: 3, under the control ofC. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselectable marker. The BnOTE protein coding sequence to express theprotein sequence given in SEQ ID NO: 29, was under the control of the P.moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitratereductase 3′UTR. The protein coding regions of BnOTE and suc2 were codonoptimized to reflect the codon bias inherent in P. moriformis UTEX 1435nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ1358 transformants of Strain A were selected on agar platescontaining sucrose as a sole carbon source, clonally purified, andsingle engineered line, Strain G was selected for analysis. Strain G wascultivated under heterotrophic lipid production conditions at pH 7.0 (toactivate expression of BnOTE from the PmAmt03 promoter) as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Oil samples obtained fromStrain A and Strain G were analyzed for fatty acid composition usingmethods described in Examples 1 and 2, and, using the methods describedin Example 8, for the regiospecificity of triacylglycerides in the oil.Fatty acid profiles of TAGs isolated from Strain A and G are shown inTable 17. Table 18 presents the regiospecificity profile of POP, POS,and SOS TAGs present in oil samples from Strain A and G.

TABLE 17 Effect of BnOTE expression on the fatty acid composition andthe sn-2 profile of TAGs produced from transformed Protothecamoriformis. Strain G Area % Strain A (pSZ1358) Fatty acid FA profile FAprofile C10:0 n.r. 0.5 C12:0 n.r. 0.5 C14:0 1.0 1.3 C16:0 23.9 25.8C18:0 3.7 30.4 C18:1 64.3 30.2 C18:2 4.5 8.8 C18:3 α n.r. 0.4

TABLE 18 Effect of BnOTE expression on the regiospecific profile of POP,POS, and SOS TAGs produced from transformed Prototheca moriformis.Strain A Strain G Cocoa (untransformed) (pSZ1358) Butter Normal- Normal-Normal- ized ized ized TAG Area % Area % Area % Area % Area % Area % POP13.09 76.8 10.6 23.5 17.9 22.1 POS 3.51 20.5 21.0 46.6 39.2 48.4 SOS0.45 2.6 13.5 29.9 23.9 29.5 total 17.05 100 45.0 100 81.1 100

As shown in Table 17, the fatty acid composition of TAGs isolated fromStrain G expressing BnOTE was markedly increased for C18:0 fatty acids(from 3.7% to 30.4%) and decreased in C18:1 fatty acids (from 64.3% to30.2%) relative to the fatty acid profile of TAGs isolated fromuntransformed Strain A. The fatty acid composition of TAGs isolated fromStrain A was characterized by about 23.9% palmitic acid, 3.7% stearicacid, and 64.3% oleic acid, a ratio for P:S:O of about 6.5:1:17.4. Incontrast, the fatty acid composition of TAGs isolated from Strain G wascharacterized by about 25.8% palmitic acid, 30.4% stearic acid, and30.2% oleic acid, a ratio for P:O:S of about 1:1.18:1.17.

The impact of expression of a C18:0 preferring thioesterease on theregiospecific profile of POP, POS, and SOS TAGs of oils produced fromthe transformed microorganism was an increase in all three TAGs as aproportion of the total TAGs present in the oil. As shown in Table 18,the sum of POP+POS+SOS TAGs accounted for 45% of the TAGs produced byStrain G, whereas POP, POS, and SOS TAGs summed to only about 17% ofTAGs produced in Strain A. The percentages of POP, POS and SOS of strainG are compared to Cocoa butter in Table 18. As can be seen, ratios ofPOP, POS and SOS of Strain G are very similar to the ratios observed incocoa butter.

These data demonstrate the utility and effectiveness of polynucleotidespermitting exogenous thioesterase expression to alter the fatty acid andregiospecific profiles of TAGs of engineered microorganisms, inparticular to increase the distribution of POP, POS, and SOS TAGs.

Examples 10-33 Engineering of Microorganisms

Examples 10-33 below describe the engineering of various microorganismsin accordance with the present invention. To alter the fatty acidprofile of a microorganism, microorganisms can be genetically modifiedwherein endogenous or exogenous lipid biosynthesis pathway enzymes areexpressed, overexpressed, or attenuated. Steps to genetically engineer amicrobe to alter its fatty acid profile as to the degree of fatty acidunsaturation and to decrease or increase fatty acid chain lengthcomprise the design and construction of a transformation vector (e.g., aplasmid), transformation of the microbe with one or more vectors,selection of transformed microbes (transformants), growth of thetransformed microbe, and analysis of the fatty acid profile of thelipids produced by the engineered microbe.

Transgenes that alter the fatty acid profiles of host organisms can beexpressed in numerous eukaryotic microbes. Examples of expression oftransgenes in eukaryotic microbes including Chlamydomonas reinhardtii,Chlorella ellipsoidea, Chlorella saccarophila, Chlorella vulgaris,Chlorella kessleri, Chlorella sorokiniana, Haematococcus pluvialis,Gonium pectorals, Volvox carteri, Dunaliella tertiolecta, Dunaliellaviridis, Dunaliella salina, Closterium peracerosum-strigosum-littoralecomplex, Nannochloropsis sp., Thalassiosira pseudonana, Phaeodactylumtricornutum, Navicula saprophila, Cylindrotheca fusiformis, Cyclotellacryptica, Symbiodinium microadriacticum, Amphidinium sp., Chaetocerossp., Mortierella alpina, and Yarrowia lipolytica can be found in thescientific literature. These expression techniques can be combined withthe teachings of the present invention to produce engineeredmicroorganisms with altered fatty acid profiles.

Transgenes that alter the fatty acid profiles of host organisms or alterthe regiospecific distribution of glycerolipids produced by hostorganisms can also be expressed in numerous prokaryotic microbes.Examples of expression of transgenes in oleaginous microbes includingRhodococcus opacus can be found in the literature. These expressiontechniques can be combined with the teachings of the present inventionto produce engineered microorganisms with altered fatty acid profiles.

TABLES 19A-D Codon preference listing. Amino Chlorella ChlorellaChlorella Chlorella Dunaliella Volvox Haematococcus Acid Codonsorokiniana vulgaris ellipsoidea kessleri tertiolecta carteri pluvialisAla GCG 0.20 0.25 0.15 0.14 0.09 0.25 0.21 Ala GCA 0.05 0.24 0.32 0.100.17 0.13 0.27 Ala GCT 0.12 0.16 0.26 0.18 0.31 0.26 0.17 Ala GCC 0.630.35 0.27 0.58 0.43 0.36 0.35 Arg AG G 0.03 0.09 0.10 0.09 0.26 0.080.14 Arg AGA 0.04 0.05 0.14 0.01 0.09 0.03 0.05 Arg CGG 0.06 0.19 0.090.06 0.06 0.17 0.15 Arg CGA 0.00 0.10 0.08 0.00 0.08 0.08 0.10 Arg CGT0.06 0.09 0.37 0.14 0.12 0.22 0.13 Arg CGC 0.81 0.48 0.22 0.71 0.40 0.430.42 Asn AAT 0.04 0.16 0.43 0.06 0.27 0.23 0.21 Asn AAC 0.96 0.84 0.570.94 0.73 0.77 0.79 Asp GAT 0.13 0.25 0.47 0.12 0.40 0.35 0.27 Asp GAC0.87 0.75 0.53 0.88 0.60 0.65 0.73 Cys TGT 0.06 0.13 0.43 0.09 0.20 0.170.27 Cys TGC 0.94 0.87 0.57 0.91 0.80 0.83 0.64 End TGA 0.00 0.72 0.140.14 0.36 0.24 0.70 End TAG 0.33 0.11 0.29 0.00 0.00 0.18 0.22 End TAA0.67 0.17 4.00 0.86 0.64 0.59 0.09 Gln CAG 0.42 0.40 0.15 0.40 0.27 0.290.33 Gln CAA 0.04 0.04 0.21 0.40 0.27 0.07 0.10 Glu GAG 0.53 0.50 0.330.40 0.27 0.53 0.49 Glu GAA 0.02 0.06 0.31 0.40 0.27 0.11 0.07 Gly GGG0.04 0.16 0.19 0.08 0.10 0.12 0.22 Gly GGA 0.02 0.11 0.13 0.07 0.13 0.120.11 Gly GGT 0.03 0.12 0.39 0.24 0.25 0.23 0.15 Gly GGC 0.91 0.61 0.290.96 0.51 0.53 0.52 His CAT 0.14 0.16 0.30 0.08 0.25 0.35 0.27 His CAC0.86 0.84 0.70 0.93 0.75 0.65 0.73 Ile ATA 0.00 0.04 0.07 0.01 0.04 0.080.09 Ile ATT 0.15 0.30 0.63 0.29 0.31 0.35 0.29 Ile ATC 0.85 0.66 0.650.69 0.65 0.57 0.62 Leu TTG 0.03 0.07 0.03 0.05 0.14 0.14 0.16 Leu TTA0.00 0.01 0.32 0.00 0.02 0.03 0.02 Leu CTG 0.72 0.61 0.34 0.61 0.60 0.450.53 Leu CTA 0.01 0.03 0.03 0.04 0.04 0.07 0.07 Leu CTT 0.04 0.08 0.160.06 0.06 0.14 0.09 Leu CTC 0.20 0.20 0.12 0.24 0.14 0.17 0.13 Lys AAG0.98 0.94 0.54 0.98 0.90 0.90 0.84 Lys AAA 0.02 0.06 0.46 0.02 0.10 0.100.16 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.28 0.32 0.420.31 0.24 0.27 0.35 Phe TTC 0.72 0.68 0.58 0.69 0.76 0.73 0.65 Pro CCG0.18 0.31 0.09 0.07 0.04 0.34 0.15 Pro CCA 0.06 0.17 0.36 0.07 0.04 0.200.24 Pro CCT 0.10 0.14 0.25 0.17 0.04 0.19 0.29 Pro CCC 0.66 0.38 0.290.69 0.04 0.27 0.32 Ser AGT 0.03 0.04 0.14 0.02 0.08 0.08 0.07 Ser AGC0.27 0.38 0.18 0.18 0.31 0.27 0.31 Ser TCG 0.12 0.14 0.08 0.10 0.02 0.190.10 Ser TCA 0.03 0.08 0.14 0.08 0.09 0.09 0.14 Ser TCT 0.09 0.11 0.260.18 0.19 0.14 0.13 Ser TCC 0.47 0.24 0.20 0.44 0.30 0.24 0.24 Thr ACG0.11 0.20 0.13 0.05 0.12 0.27 0.19 Thr ACA 0.01 0.20 0.32 0.07 0.20 0.120.23 Thr ACT 0.12 0.13 0.29 0.12 0.24 0.20 0.18 Thr ACC 0.76 0.47 0.260.76 0.44 0.41 0.40 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT0.07 0.15 0.43 0.27 0.28 0.24 0.19 Tyr TAC 0.93 0.85 0.57 0.73 0.72 0.760.81 Val GTG 0.71 0.54 0.37 0.60 0.54 0.46 0.62 Val GTA 0.00 0.05 0.250.03 0.09 0.07 0.09 Val GTT 0.11 0.14 0.24 0.09 0.14 0.17 0.09 Val GTC0.18 0.27 0.14 0.28 0.23 0.30 0.21 Closterium peracerosum- strigosum-Amino littorale Dunaliella Dunaliella Gonium Phaeodactylum ChaetocerosAcid Codon complex viridis salina pectorale tricornutum compressum AlaGCG 0.48 0.13 0.15 0.43 0.15 0.08 Ala GCA 0.10 0.27 0.20 0.09 0.10 0.37Ala GCT 0.15 0.25 0.27 0.08 0.23 0.36 Ala GCC 0.26 0.35 0.39 0.41 0.520.18 Arg AGG 0.04 0.25 0.22 0.13 0.02 0.14 Arg AGA 0.00 0.06 0.05 0.000.04 0.29 Arg CGG 0.18 0.08 0.12 0.40 0.10 0.00 Arg CGA 0.00 0.06 0.060.05 0.12 0.19 Arg CGT 0.13 0.15 0.13 0.08 0.41 0.38 Arg CGC 0.64 0.390.43 0.35 0.31 0.00 Asn AAT 0.04 0.17 0.23 0.07 0.30 0.58 Asn AAC 0.960.83 0.77 0.93 0.65 0.42 Asp GAT 0.30 0.38 0.40 0.11 0.41 0.53 Asp GAC0.70 0.62 0.60 0.89 0.59 0.47 Cys TGT 0.06 0.24 0.17 0.20 0.39 0.44 CysTGC 0.94 0.76 0.83 0.90 0.61 0.56 End TGA 0.75 0.31 0.37 0.50 0.06 0.50End TAG 0.00 0.15 0.14 0.00 0.13 0.00 End TAA 0.25 0.54 0.49 0.50 0.810.50 Gln CAG 0.53 0.36 0.32 0.31 0.23 0.16 Gln CAA 0.09 0.12 0.08 0.070.14 0.19 Glu GAG 0.31 0.44 0.51 0.56 0.21 0.28 Glu GAA 0.06 0.09 0.090.07 0.42 0.37 Gly GGG 0.31 0.14 0.10 0.18 0.08 0.12 Gly GGA 0.06 0.110.12 0.09 0.34 0.33 Gly GGT 0.09 0.22 0.22 0.07 0.30 0.39 Gly GGC 0.530.54 0.56 0.65 0.28 0.16 His CAT 0.33 0.25 0.25 0.43 0.28 0.84 His CAC0.67 0.75 0.75 0.57 0.72 0.16 Ile ATA 0.03 0.03 0.03 0.07 0.03 0.12 IleATT 0.23 0.25 0.31 0.33 0.51 0.65 Ile ATC 0.74 0.72 0.66 0.59 0.46 0.23Leu TTG 0.04 0.11 0.12 0.04 0.26 0.11 Leu TTA 0.00 0.01 0.01 0.00 0.020.14 Leu CTG 0.31 0.60 0.61 0.64 0.15 0.05 Leu CTA 0.01 0.05 0.04 0.010.05 0.08 Leu CTT 0.04 0.07 0.08 0.05 0.18 0.51 Leu CTC 0.60 0.16 0.140.26 0.34 0.11 Lys AAG 0.86 0.87 0.89 0.93 0.75 0.52 Lys AAA 0.14 0.130.11 0.07 0.25 0.48 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.090.25 0.29 0.10 0.44 0.65 Phe TTC 0.91 0.75 0.71 0.90 0.56 0.35 Pro CCG0.28 0.10 0.08 0.53 0.29 0.05 Pro CCA 0.15 0.10 0.17 0.09 0.12 0.45 ProCCT 0.12 0.10 0.30 0.04 0.20 0.33 Pro CCC 0.44 0.10 0.45 0.34 0.40 0.17Ser AGT 0.04 0.09 0.06 0.02 0.12 0.14 Ser AGC 0.05 0.31 0.32 0.20 0.120.07 Ser TCG 0.22 0.04 0.06 0.42 0.19 0.08 Ser TCA 0.16 0.08 0.10 0.090.06 0.31 Ser TCT 0.05 0.17 0.15 0.07 0.15 0.23 Ser TCC 0.47 0.31 0.300.20 0.35 0.18 Thr ACG 0.30 0.16 0.13 0.42 0.23 0.10 Thr ACA 0.06 0.210.18 0.03 0.13 0.38 Thr ACT 0.22 0.18 0.23 0.08 0.19 0.27 Thr ACC 0.420.46 0.46 0.47 0.45 0.25 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT0.07 0.16 0.21 0.12 0.18 0.67 Tyr TAC 0.93 0.84 0.79 0.88 0.82 0.33 ValGTG 0.50 0.64 0.62 0.57 0.22 0.30 Val GTA 0.02 0.03 0.05 0.04 0.09 0.27Val GTT 0.06 0.11 0.11 0.04 0.22 0.10 Val GTC 0.42 0.22 0.23 0.35 0.470.33 Symbio- Cylindro- Amphi- dinium Nanno- Amino theca dinium micro-chloropsis Cyclotella Navicula Thalassiosira C. Acid Codon fusiformiscarterae adriacticum sp cryptica pelliculosa pseudonana reinhardtii AlaGCG 0.07 0.17 0.22 0.24 0.11 0.00 0.11 0.35 Ala GCA 0.14 0.33 0.26 0.100.16 0.13 0.25 0.08 Ala GCT 0.35 0.29 0.20 0.17 0.45 0.44 0.33 0.13 AlaGCC 0.43 0.20 0.32 0.48 0.27 0.44 0.30 0.43 Arg AGG 0.09 0.15 0.27 0.000.09 0.05 0.18 0.05 Arg AGA 0.14 0.03 0.27 0.00 0.05 0.10 0.17 0.01 ArgCGG 0.06 0.08 0.09 0.00 0.04 0.05 0.06 0.20 Arg CGA 0.16 0.18 0.09 0.290.08 0.35 0.11 0.04 Arg CGT 0.34 0.18 0.09 0.14 0.47 0.20 0.34 0.09 ArgCGC 0.22 0.40 0.18 0.57 0.28 0.25 0.15 0.62 Asn AAT 0.42 0.37 0.21 0.000.25 0.47 0.43 0.09 Asn AAC 0.58 0.63 0.79 1.00 0.75 0.53 0.57 0.91 AspGAT 0.54 0.54 0.50 0.20 0.52 0.20 0.56 0.14 Asp GAC 0.46 0.46 0.50 0.800.48 0.80 0.44 0.86 Cys TGT 0.44 0.75 0.50 0.00 0.29 0.10 0.54 0.10 CysTGC 0.56 0.25 0.50 1.00 0.71 0.90 0.46 0.90 End TGA 0.13 0.50 1.00 0.000.10 0.00 0.31 0.27 End TAG 0.10 0.00 0.00 0.00 0.00 0.00 0.38 0.22 EndTAA 0.77 0.50 0.00 1.00 0.90 1.00 0.31 0.52 Gln CAG 0.12 0.33 0.28 0.410.19 0.21 0.16 0.38 Gln CAA 0.25 0.15 0.17 0.00 0.17 0.28 0.19 0.04 GluGAG 0.23 0.41 0.50 0.59 0.38 0.17 0.40 0.55 Glu GAA 0.39 0.10 0.06 0.000.26 0.34 0.26 0.03 Gly GGG 0.06 0.19 0.32 0.10 0.10 0.03 0.12 0.11 GlyGGA 0.47 0.10 0.12 0.05 0.45 0.28 0.51 0.06 Gly GGT 0.35 0.34 0.16 0.250.22 0.13 0.23 0.11 Gly GGC 0.12 0.37 0.40 0.60 0.24 0.56 0.14 0.72 HisCAT 0.39 0.12 0.40 0.00 0.42 1.00 0.50 0.11 His CAC 0.61 0.88 0.60 1.000.58 0.00 0.50 0.89 Ile ATA 0.06 0.05 0.00 0.00 0.04 0.00 0.08 0.03 IleATT 0.42 0.53 0.38 0.14 0.53 0.73 0.38 0.22 Ile ATC 0.52 0.42 0.63 0.860.42 0.27 0.54 0.75 Leu TTG 0.26 0.35 0.39 0.22 0.20 0.16 0.29 0.04 LeuTTA 0.09 0.01 0.00 0.00 0.03 0.00 0.05 0.01 Leu CTG 0.09 0.22 0.39 0.090.06 0.12 0.08 0.73 Leu CTA 0.05 0.00 0.04 0.00 0.03 0.04 0.06 0.03 LeuCTT 0.37 0.31 0.13 0.04 0.39 0.36 0.20 0.05 Leu CTC 0.13 0.12 0.04 0.650.29 0.32 0.32 0.15 Lys AAG 0.60 0.93 0.85 1.00 0.70 0.83 0.76 0.95 LysAAA 0.40 0.07 0.15 0.00 0.30 0.17 0.24 0.05 Met ATG 1.00 1.00 1.00 1.001.00 1.00 1.00 1.00 Phe TTT 0.37 0.21 0.25 0.20 0.31 0.78 0.38 0.16 PheTTC 0.63 0.79 0.75 0.80 0.69 0.22 0.62 0.84 Pro CCG 0.11 0.14 0.18 0.080.10 0.21 0.16 0.33 Pro CCA 0.33 0.42 0.09 0.08 0.16 0.29 0.31 0.08 ProCCT 0.32 0.22 0.41 0.25 0.35 0.21 0.31 0.13 Pro CCC 0.24 0.22 0.32 0.580.39 0.29 0.23 0.47 Ser AGT 0.12 0.13 0.09 0.00 0.09 0.13 0.18 0.04 SerAGC 0.09 0.24 0.14 0.13 0.08 0.28 0.11 0.35 Ser TCG 0.13 0.03 0.05 0.000.15 0.25 0.17 0.25 Ser TCA 0.12 0.25 0.05 0.00 0.12 0.08 0.12 0.05 SerTCT 0.30 0.16 0.23 0.13 0.39 0.25 0.23 0.07 Ser TCC 0.24 0.19 0.45 0.750.18 0.03 0.19 0.25 Thr ACG 0.09 0.14 0.10 0.28 0.10 0.18 0.21 0.30 ThrACA 0.15 0.28 0.10 0.00 0.15 0.09 0.19 0.08 Thr ACT 0.39 0.12 0.10 0.170.33 0.41 0.28 0.10 Thr ACC 0.37 0.47 0.70 0.56 0.43 0.32 0.32 0.52 TrpTGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.38 0.32 0.20 0.000.38 0.20 0.39 0.10 Tyr TAC 0.62 0.68 0.80 1.00 0.62 0.80 0.61 0.90 ValGTG 0.11 0.65 0.67 0.31 0.16 0.18 0.29 0.67 Val GTA 0.06 0.05 0.00 0.000.09 0.09 0.16 0.03 Val GTT 0.38 0.08 0.11 0.15 0.42 0.09 0.28 0.07 ValGTC 0.46 0.21 0.22 0.54 0.33 0.64 0.27 0.22 Amino Yarrowia MortierellaRhodococcus Acid Codon lipolytica alpina opacus Ala GCG 0.08 0.14 0.35Ala GCA 0.11 0.12 0.14 Ala GCT 0.35 0.29 0.09 Ala GCC 0.46 0.45 0.43 ArgAGG 0.05 0.05 0.05 Arg AGA 0.13 0.06 0.02 Arg CGG 0.12 0.06 0.26 Arg CGA0.52 0.09 0.12 Arg CGT 0.11 0.32 0.11 Arg CGC 0.07 0.42 0.44 Asn AAT0.17 0.15 0.21 Asn AAC 0.83 0.85 0.79 Asp GAT 0.35 0.42 0.24 Asp GAC0.65 0.58 0.76 Cys TGT 0.46 0.13 0.26 Cys TGC 0.54 0.87 0.74 End TGA0.16 0.05 0.72 End TAG 0.38 0.25 0.17 End TAA 0.46 0.70 0.11 Gln CAG0.33 0.36 0.28 Gln CAA 0.08 0.06 0.06 Glu GAG 0.44 0.49 0.45 Glu GAA0.14 0.09 0.22 Gly GGG 0.05 0.03 0.18 Gly GGA 0.28 0.29 0.15 Gly GGT0.32 0.32 0.20 Gly GGC 0.34 0.36 0.48 His CAT 0.34 0.27 0.20 His CAC0.66 0.73 0.80 Ile ATA 0.03 0.01 0.05 Ile ATT 0.44 0.33 0.14 Ile ATC0.53 0.66 0.81 Leu TTG 0.09 0.27 0.09 Leu TTA 0.02 0.00 0.01 Leu CTG0.37 0.26 0.41 Leu CTA 0.05 0.02 0.03 Leu CTT 0.18 0.12 0.06 Leu CTC0.29 0.32 0.40 Lys AAG 0.84 0.91 0.80 Lys AAA 0.16 0.09 0.20 Met ATG1.00 1.00 1.00 Phe TTT 0.38 0.39 0.09 Phe TTC 0.62 0.61 0.91 Pro CCG0.10 0.07 0.52 Pro CCA 0.10 0.08 0.09 Pro CCT 0.32 0.36 0.07 Pro CCC0.47 0.49 0.32 Ser AGT 0.07 0.05 0.08 Ser AGC 0.11 0.14 0.23 Ser TCG0.16 0.32 0.33 Ser TCA 0.08 0.08 0.07 Ser TCT 0.28 0.12 0.05 Ser TCC0.30 0.29 0.24 Thr ACG 0.11 0.17 0.28 Thr ACA 0.14 0.10 0.11 Thr ACT0.26 0.23 0.07 Thr ACC 0.49 0.49 0.53 Trp TGG 1.00 1.00 1.00 Tyr TAT0.18 0.20 0.18 Tyr TAC 0.82 0.80 0.82 Val GTG 0.33 0.22 0.37 Val GTA0.05 0.02 0.05 Val GTT 0.26 0.27 0.10 Val GTC 0.36 0.49 0.49

3-Ketoacyl ACP synthase Cuphea hookeriana 3-ketoacyl-ACP synthase(GenBank Acc. No. AAC68861.1), Cuphea wrightii beta-ketoacyl-ACPsynthase II (GenBank Acc. No. AAB37271.1), Cuphea lanceolatabeta-ketoacyl-ACP synthase IV (GenBank Acc. No. CAC59946.1), Cupheawrightii beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAB37270.1),Ricinus communis ketoacyl-ACP synthase (GenBank Acc. No. XP_002516228),Gossypium hirsutum ketoacyl- ACP synthase (GenBank Acc. No. ADK23940.1),Glycine max plastid 3-keto-acyl-ACP synthase II-A (GenBank Acc No.AAW88763.1), Elaeis guineensis beta-ketoacyl-ACP synthase II (GenBankAcc. No. AAF26738.2), Helianthus annuus plastid 3-keto-acyl-ACP synthaseI (GenkBank Acc. No. ABM53471.1), Glycine max3-keto-acyl-ACP synthase I(GenBank Acc. No. NP_001238610.1), Helianthus annuus plastid3-keto-acyl-ACP synthase II (GenBank Acc ABI18155.1), Brassica napusbeta-ketoacyl-ACP synthetase 2 (GenBank Acc. No. AAF61739.1), Perillafrutescens beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAC04692.1),Helianthus annus beta-ketoacyl-ACP synthase II (GenBank Accession No.ABI18155), Ricinus communis beta-ketoacyl-ACP synthase II (GenBankAccession No. AAA33872), Haematococcus pluvialis beta-ketoacyl acylcarrier protein synthase (GenBank Accession No. HM560033.1), Jatrophacurcasbeta ketoacyl-ACP synthase I (GenBank Accession No. ABJ90468.1),Populus trichocarpa beta-ketoacyl-ACP synthase I (GenBank Accession No.XP_002303661.1), Coriandrum sativum beta-ketoacyl-ACP synthetase I(GenBank Accession No. AAK58535.1), Arabidopsis thaliana3-oxoacyl-[acyl-carrier- protein] synthase I (GenBank Accession No.NP_001190479.1), Vitis vinifera 3-oxoacyl- [acyl-carrier-protein]synthase I (GenBank Accession No. XP_002272874.2) Fatty acyl-ACPThioesterases Umbellularia californica fatty acyl-ACP thioesterase(GenBank Acc. No. AAC49001), Cinnamomum camphora fatty acyl-ACPthioesterase (GenBank Acc. No. Q39473), Umbellularia californica fattyacyl-ACP thioesterase (GenBank Acc. No. Q41635), Myristica fragransfatty acyl-ACP thioesterase (GenBank Acc. No. AAB71729), Myristicafragrans fatty acyl-ACP thioesterase (GenBank Acc. No. AAB71730), Elaeisguineensis fatty acyl- ACP thioesterase (GenBank Acc. No. ABD83939),Elaeis guineensis fatty acyl-ACP thioesterase (GenBank Acc. No.AAD42220), Populus tomentosa fatty acyl-ACP thioesterase (GenBank Acc.No. ABC47311), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBankAcc. No. NP_172327), Arabidopsis thaliana fatty acyl-ACP thioesterase(GenBank Acc. No. CAA85387), Arabidopsis thaliana fatty acyl-ACPthioesterase (GenBank Acc. No. CAA85388), Gossypium hirsutum fattyacyl-ACP thioesterase (GenBank Acc. No. Q9SQI3), Cuphea lanceolata fattyacyl-ACP thioesterase (GenBank Acc. No. CAA54060), Cuphea hookerianafatty acyl-ACP thioesterase (GenBank Acc. No. AAC72882), Cupheacalophylla subsp. mesostemon fatty acyl-ACP thioesterase (GenBank Acc.No. ABB71581), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBankAcc. No. CAC19933), Elaeis guineensis fatty acyl-ACP thioesterase(GenBank Acc. No. AAL15645), Cuphea hookeriana fatty acyl- ACPthioesterase (GenBank Acc. No. Q39513), Gossypium hirsutum fattyacyl-ACP thioesterase (GenBank Acc. No. AAD01982), Vitis vinifera fattyacyl-ACP thioesterase (GenBank Acc. No. CAN81819), Garcinia mangostanafatty acyl-ACP thioesterase (GenBank Acc. No. AAB51525), Brassica junceafatty acyl-ACP thioesterase (GenBank Acc. No. ABI18986), Madhucalongifolia fatty acyl-ACP thioesterase (GenBank Acc. No. AAX51637),Brassica napus fatty acyl-ACP thioesterase (GenBank Acc. No. ABH11710),Brassica napus fatty acyl-ACP thioesterase (GenBank Acc. No.CAA52070.1), Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank Acc. No. EAY86877), Oryza sativa (japonicacultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No.NP_001068400), Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank Acc. No. EAY99617), Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank Acc. No. AAC49269), Ulmus Americana fattyacyl-ACP thioesterase (GenBank Acc. No. AAB71731), Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank Acc. No. CAB60830), Cupheapalustris fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49180), Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858, Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858.1),Cuphea palustris fatty acyl-ACP thioesterase (GenBank Acc. No.AAC49179), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc.No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBankAcc. No. AAB717291.1), Cuphea hookeriana fatty acyl-ACP thioesteraseGenBank Acc. No. U39834), Umbelluaria californica fatty acyl-ACPthioesterase (GenBank Acc. No. M94159), Cinnamomum camphora fattyacyl-ACP thioesterase (GenBank Acc. No. U31813), Ricinus communis fattyacyl-ACP thioesterase (GenBank Acc. No. ABS30422.1), Helianthus annuusacyl-ACP thioesterase (GenBank Accession No. AAL79361.1), Jatrophacurcas acyl-ACP thioesterase (GenBank Accession No. ABX82799.3), Zeamays oleoyl-acyl carrier protein thioesterase, (GenBank Accession No.ACG40089.1), Haematococcus pluvialis fatty acyl- ACP thioesterase(GenBank Accession No. HM560034.1) Desaturase Enzymes Linumusitatissimum fatty acid desaturase 3C, (GenBank Acc. No. ADV92272.1),Ricinus communis omega-3 fatty acid desaturase, endoplasmic reticulum,putative, (GenBank Acc. No. EEF36775.1), Vernicia fordii omega-3 fattyacid desaturase, (GenBank Acc. No. AAF12821), Glycine max chloroplastomega 3 fatty acid desaturase isoform 2, (GenBank Acc. No. ACF19424.1),Prototheca moriformis FAD-D omega 3 desaturase (SEQ ID NO: 35),Prototheca moriformis linoleate desaturase (SEQ ID NO: 36), Carthamustinctorius delta 12 desaturase, (GenBank Accession No. ADM48790.1),Gossypium hirsutum omega-6 desaturase, (GenBank Accession No.CAA71199.1), Glycine max microsomal desaturase (GenBank Accession No.BAD89862.1), Zea mays fatty acid desaturase (GenBank Accession No.ABF50053.1), Brassica napa linoleic acid desaturase (GenBank AccessionNo. AAA32994.1), Camelina sativa omega-3 desaturase (SEQ ID NO: 37),Prototheca moriformis delta 12 desaturase allele 2 (SEQ ID NO: 38,Camelina sativa omega-3 FAD7-1 (SEQ ID NO: 39), Helianthus annuusstearoyl-ACP desaturase, (GenBank Accession No. AAB65145.1), Ricinuscommunis stearoyl-ACP desaturase, (GenBank Accession No. AACG59946.1),Brassica juncea plastidic delta-9-stearoyl-ACP desaturase (GenBankAccession No. AAD40245.1), Glycine max stearoyl-ACP desaturase (GenBankAccession No. ACJ39209.1), Olea europaea stearoyl-ACP desaturase(GenBank Accession No. AAB67840.1), Vernicia fordiistearoyl-acyl-carrier protein desaturase, (GenBank Accession No.ADC32803.1), Descurainia sophia delta-12 fatty acid desaturase (GenBankAccession No. ABS86964.2), Euphorbia lagascae delta12-oleic aciddesaturase (GenBank Acc. No. AAS57577.1), Chlorella vulgaris delta 12fatty acid desaturase (GenBank Accession No. ACF98528), Chlorellavulgaris omega-3 fatty acid desaturase (GenBank Accession No. BAB78717),Haematococcus pluvialis omega-3 fatty acid desaturase (GenBank AccessionNo. HM560035.1), Haematococcus pluvialis stearoyl-ACP-desaturase GenBankAccession No. EF586860.1, Haematococcus pluvialisstearoyl-ACP-desaturase GenBank Accession No. EF523479.1 Oleate12-hydroxylase Enzymes Ricinus communis oleate 12-hydroxylase (GenBankAcc. No. AAC49010.1), Physaria lindheimeri oleate 12-hydroxylase(GenBank Acc. No. ABQ01458.1), Physaria lindheimeri mutant bifunctionaloleate 12-hydroxylase: desaturase (GenBank Acc. No. ACF17571.1),Physaria lindheimeri bifunctional oleate 12-hydroxylase: desaturase(GenBank Accession No. ACQ42234.1), Physaria lindheimeri bifunctionaloleate 12- hydroxylase: desaturase (GenBank Acc. No. AAC32755.1),Arabidopsis lyrata subsp. Lyrata (GenBank Acc. No. XP_002884883.1)Glycerol-3-phosphate Enzymes Arabidopsis thaliana glycerol-3-phosphateacyltransferase BAA00575, Chlamydomonas reinhardtii glycerol-3-phosphateacyltransferase (GenBank Acc. No. EDP02129), Chlamydomonas reinhardtiiglycerol-3-phosphate acyltransferase (GenBank Acc. No. Q886Q7),Cucurbita moschata acyl-(acyl-carrier-protein): glycerol-3-phosphateacyltransferase (GenBank Acc. No. BAB39688), Elaeis guineensisglycerol-3-phosphate acyltransferase, ((GenBank Acc. No. AAF64066),Garcina mangostana glycerol-3-phosphate acyltransferase (GenBank Acc.No. ABS86942), Gossypium hirsutum glycerol-3-phosphate acyltransferase(GenBank Acc. No. ADK23938), Jatropha curcas glycerol-3-phosphateacyltransferase (GenBank Acc. No. ADV77219), Jatropha curcas plastidglycerol-3- phosphate acyltransferase (GenBank Acc. No. ACR61638),Ricinus communis plastidial glycerol-phosphate acyltransferase (GenBankAcc. No. EEF43526), Vica faba glycerol-3- phosphate acyltransferase(GenBank Accession No. AAD05164), Zea mays glycerol-3- phosphateacyltransferase (GenBank Acc. No. ACG45812) Lysophosphatidic acidacyltransferase Enzymes Arabidopsis thaliana1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.AEE85783), Brassica juncea 1-acyl-sn-glycerol-3-phosphateacyltransferase (GenBank Accession No. ABQ42862), Brassica juncea1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.ABM92334), Brassica napus 1-acyl-sn-glycerol-3-phosphate acyltransferase(GenBank Accession No. CAB09138), Chlamydomonas reinhardtiilysophosphatidic acid acyltransferase (GenBank Accession No. EDP02300),Cocos nucifera lysophosphatidic acid acyltransferase (GenBank Acc. No.AAC49119), Limnanthes alba lysophosphatidic acid acyltransferase(GenBank Accession No. EDP02300), Limnanthes douglasii1-acyl-sn-glycerol-3-phosphate acyltransferase (putative) (GenBankAccession No. CAA88620), Limnanthes douglasii acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (GenBank Accession No.ABD62751), Limnanthes douglasii 1-acylglycerol-3-phosphate O-acyltransferase (GenBank Accession No. CAA58239), Ricinus communis1-acyl-sn-glycerol- 3-phosphate acyltransferase (GenBank Accession No.EEF39377) Diacylglycerol acyltransferase Enzymes Arabidopsis thalianadiacylglycerol acyltransferase (GenBank Acc. No. CAB45373), Brassicajuncea diacylglycerol acyltransferase (GenBank Acc. No. AAY40784),Elaeis guineensis putative diacylglycerol acyltransferase (GenBank Acc.No. AEQ94187), Elaeis guineensis putative diacylglycerol acyltransferase(GenBank Acc. No. AEQ94186), Glycine max acyl CoA: diacylglycerolacyltransferase (GenBank Acc. No. AAT73629), Helianthus annusdiacylglycerol acyltransferase (GenBank Acc. No. ABX61081), Oleaeuropaea acyl- CoA: diacylglycerol acyltransferase 1 (GenBank Acc. No.AAS01606), Ricinus communis diacylglycerol acyltransferase (GenBank Acc.No. AAR11479) Phospholipid diacylglycerol acyltransferase EnzymesArabidopsis thaliana phospholipid: diacylglycerol acyltransferase(GenBank Acc. No. AED91921), Elaeis guineensis putative phospholipid:diacylglycerol acyltransferase (GenBank Acc. No. AEQ94116), Glycine maxphospholipid: diacylglycerol acyltransferase 1-like (GenBank Acc. No.XP_003541296), Jatropha curcas phospholipid: diacylglycerolacyltransferase (GenBank Acc. No. AEZ56255), Ricinus communisphospholipid: diacylglycerol acyltransferase (GenBank Acc. No.ADK92410), Ricinus communis phospholipid: diacylglycerol acyltransferase(GenBank Acc. No. AEW99982)

Example 10 Engineering Chlorella sorokiniana

Expression of recombinant genes in accordance with the present inventionin Chlorella sorokiniana can be accomplished by modifying the methodsand vectors taught by Dawson et al. as discussed herein. Briefly, Dawsonet al., Current Microbiology Vol. 35 (1997) pp. 356-362, reported thestable nuclear transformation of Chlorella sorokiniana with plasmid DNA.Using the transformation method of microprojectile bombardment, Dawsonintroduced the plasmid pSV72-NRg, encoding the full Chlorella vulgarisnitrate reductase gene (NR, GenBank Accession No. U39931), into mutantChlorella sorokiniana (NR-mutants). The NR-mutants are incapable ofgrowth without the use of nitrate as a source of nitrogen. Nitratereductase catalyzes the conversion of nitrate to nitrite. Prior totransformation, Chlorella sorokiniana NR-mutants were unable to growbeyond the microcolony stage on culture medium comprising nitrate (NO₃⁻) as the sole nitrogen source. The expression of the Chlorella vulgarisNR gene product in NR-mutant Chlorella sorokiniana was used as aselectable marker to rescue the nitrate metabolism deficiency. Upontransformation with the pSV72-NRg plasmid, NR-mutant Chlorellasorokiniana stably expressing the Chlorella vulgaris NR gene productwere obtained that were able to grow beyond the microcolony stage onagar plates comprising nitrate as the sole carbon source. Evaluation ofthe DNA of the stable transformants was performed by Southern analysisand evaluation of the RNA of the stable transformants was performed byRNase protection. Selection and maintenance of the transformed Chlorellasorokiniana (NR mutant) was performed on agar plates (pH 7.4) comprising0.2 g/L MgSO₄, 0.67 g/L KH₂PO₄, 3.5 g/L K₂HPO₄, 1.0 g/L Na₃C₆H₅O₇.H₂Oand 16.0 g/L agar, an appropriate nitrogen source (e.g., NO₃ ⁻),micronutrients, and a carbon source. Dawson also reported thepropagation of Chlorella sorokiniana and Chlorella sorokiniana NRmutants in liquid culture medium. Dawson reported that the plasmidpSV72-NRg and the promoter and 3′ UTR/terminator of the Chlorellavulgaris nitrate reductase gene were suitable to enable heterologousgene expression in Chlorella sorokiniana NR-mutants. Dawson alsoreported that expression of the Chlorella vulgaris nitrate reductasegene product was suitable for use as a selectable marker in Chlorellasorokiniana NR-mutants.

In an embodiment of the present invention, vector pSV72-NRg, comprisingnucleotide sequence encoding the Chlorella vulgaris nitrate reductase(CvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellasorokiniana to reflect the codon bias inherent in nuclear genes ofChlorella sorokiniana in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the CvNR promoterupstream of the protein-coding sequence and operably linked to the CvNR3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chlorella sorokiniana genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorellasorokiniana with the transformation vector is achieved throughwell-known transformation techniques including microprojectilebombardment or other known methods. Activity of the CvNR gene productcan be used as a selectable marker to rescue the nitrogen assimilationdeficiency of Chlorella sorokiniana NR mutant strains and to select forChlorella sorokiniana NR-mutants stably expressing the transformationvector. Growth media suitable for Chlorella sorokiniana lipid productioninclude, but are not limited to 0.5 g/L KH₂PO₄, 0.5 g/L K₂HPO₄, 0.25 g/LMgSO₄-7H2O, with supplemental micronutrients and the appropriatenitrogen and carbon sources (Patterson, Lipids Vol. 5:7 (1970), pp.597-600). Evaluation of fatty acid profiles of Chlorella sorokinianalipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 11 Engineering Chlorella vulgaris

Expression of recombinant genes in accordance with the present inventionin Chlorella vulgaris can be accomplished by modifying the methods andvectors taught by Chow and Tung et al. as discussed herein. Briefly,Chow and Tung et al., Plant Cell Reports, Volume 18 (1999), pp. 778-780,reported the stable nuclear transformation of Chlorella vulgaris withplasmid DNA. Using the transformation method of electroporation, Chowand Tung introduced the plasmid pIG121-Hm (GenBank Accession No.AB489142) into Chlorella vulgaris. The nucleotide sequence of pIG121-Hmcomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter upstream of the GUSprotein-coding sequence and further operably linked to the 3′UTR/terminator of the nopaline synthase (nos) gene downstream of the GUSprotein-coding sequence. The sequence of plasmid pIG121-Hm furthercomprised a hygromycin B antibiotic resistance cassette. This hygromycinB antibiotic resistance cassette comprised a CaMV 35S promoter operablylinked to sequence encoding the hygromycin phosphotransferase (hpt,GenBank Accession No. BAH24259) gene product. Prior to transformation,Chlorella vulgaris was unable to be propagated in culture mediumcomprising 50 ug/ml hygromycin B. Upon transformation with the pIG121-Hmplasmid, transformants of Chlorella vulgaris were obtained that werepropagated in culture medium comprising 50 ug/ml hygromycin B. Theexpression of the hpt gene product in Chlorella vulgaris enabledpropagation of transformed Chlorella vulgaris in the presence of 50ug/mL hygromycin B, thereby establishing the utility of the a hygromycinB resistance cassette as a selectable marker for use in Chlorellavulgaris. Detectable activity of the GUS reporter gene indicated thatCaMV 35S promoter and nos 3′UTR are suitable for enabling heterologousgene expression in Chlorella vulgaris. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Selectionand maintenance of transformed Chlorella vulgaris was performed on agarplates comprising YA medium (agar and 4 g/L yeast extract). Thepropagation of Chlorella vulgaris in liquid culture medium was conductedas discussed by Chow and Tung. Propagation of Chlorella vulgaris inmedia other than YA medium has been described (for examples, see Chaderet al., Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26 andIllman et al., Enzyme and Microbial Technology, Vol. 27 (2000), pp.631-635). Chow and Tung reported that the plasmid pIG121-Hm, the CaMV35S promoter, and the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator are suitable to enable heterologous gene expression inChlorella vulgaris. In addition, Chow and Tung reported the hygromycin Bresistance cassette was suitable for use as a selectable marker inChlorella vulgaris. Additional plasmids, promoters, 3′UTR/terminators,and selectable markers suitable for enabling heterologous geneexpression in Chlorella vulgaris have been discussed in Chader et al.,Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26.

In an embodiment of the present invention, pIG121-Hm, comprising thenucleotide sequence encoding the hygromycin B gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Chlorella vulgaris to reflect thecodon bias inherent in nuclear genes of Chlorella vulgaris in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the CaMV 35S promoter upstream of the protein-coding sequenceand operably linked to the Agrobacterium tumefaciens nopaline synthasegene 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Chlorella vulgaris genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorella vulgariswith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the hygromycin B resistance gene product can beused as a marker to select for Chlorella vulgaris transformed with thetransformation vector on, but not limited to, agar medium comprisinghygromycin. Growth media suitable for Chlorella vulgaris lipidproduction include, but are not limited to BG11 medium (0.04 g/L KH₂PO₄,0.075 g/L CaCl₂, 0.036 g/L citric acid, 0.006 g/L Ammonium FerricCitrate, 1 mg/L EDTA, and 0.02 g/L Na₂CO₃) supplemented with tracemetals, and optionally 1.5 g/L NaNO3. Additional media suitable forculturing Chlorella vulgaris for lipid production include, for example,Watanabe medium (comprising 1.5 g/L KNO₃, 1.25 g/L KH₂PO₄, 1.25 g⁻¹MgSO₄.7H₂O, 20 mg⁻¹ FeSO₄.7H₂O with micronutrients and low-nitrogenmedium (comprising 203 mg/l (NH₄)₂HPO₄, 2.236 g/l KCl, 2.465 g/l MgSO₄,1.361 g/l KH₂PO₄ and 10 mg/l FeSO₄) as reported by Illman et al., Enzymeand Microbial Technology, Vol. 27 (2000), pp. 631-635. Evaluation offatty acid profiles of Chlorella vulgaris lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 12 Engineering Chlorella ellipsoidea

Expression of recombinant genes in accordance with the present inventionin Chlorella ellipsoidea can be accomplished by modifying the methodsand vectors taught by Chen et al. as discussed herein. Briefly, Chen etal., Current Genetics, Vol. 39:5 (2001), pp. 365-370, reported thestable transformation of Chlorella ellipsoidea with plasmid DNA. Usingthe transformation method of electroporation, Chen introduced theplasmid pBinUΩNP-1 into Chlorella ellipsoidea. The nucleotide sequenceof pBinUΩNP-1 comprised sequence encoding the neutrophil peptide-1(NP-1) rabbit gene product operably linked to a Zea mays Ubiquitin(ubi1) gene promoter upstream of the NP-1 protein-coding region andoperably linked to the 3′ UTR/terminator of the nopaline synthase (nos)gene downstream of the NP-1 protein-coding region. The sequence ofplasmid pBinUΩNP-1 further comprised a G418 antibiotic resistancecassette. This G418 antibiotic resistance cassette comprised sequenceencoding the aminoglycoside 3′-phosphotransferase (aph 3′) gene product.The aph 3′ gene product confers resistance to the antibiotic G418. Priorto transformation, Chlorella ellipsoidea was unable to be propagated inculture medium comprising 30 ug/mL G418. Upon transformation with thepBinUΩNP-1 plasmid, transformants of Chlorella ellipsoidea were obtainedthat were propagated in selective culture medium comprising 30 ug/mLG418. The expression of the aph 3′ gene product in Chlorella ellipsoideaenabled propagation of transformed Chlorella ellipsoidea in the presenceof 30 ug/mL G418, thereby establishing the utility of the G418antibiotic resistance cassette as selectable marker for use in Chlorellaellipsoidea. Detectable activity of the NP-1 gene product indicated thatthe ubi1 promoter and nos 3′ UTR are suitable for enabling heterologousgene expression in Chlorella ellipsoidea. Evaluation of the genomic DNAof the stable transformants was performed by Southern analysis.Selection and maintenance of the transformed Chlorella ellipsoidea wasperformed on Knop medium (comprising 0.2 g/L K₂HPO₄, 0.2 g/L MgSO₄.7H₂O,0.12 g/L KCl, and 10 mg/L FeCl3, pH 6.0-8.0 supplemented with 0.1% yeastextract and 0.2% glucose) with 15 ug/mL G418 (for liquid cultures) orwith 30 ug/mL G418 (for solid cultures comprising 1.8% agar).Propagation of Chlorella ellipsoidea in media other than Knop medium hasbeen reported (see Cho et al., Fisheries Science, Vol. 73:5 (2007), pp.1050-1056, Jarvis and Brown, Current Genetics, Vol. 19 (1991), pp.317-321 and Kim et al., Marine Biotechnology, Vol. 4 (2002), pp. 63-73).Additional plasmids, promoters, 3′UTR/terminators, and selectablemarkers suitable for enabling heterologous gene expression in Chlorellaellipsoidea have been reported (see Jarvis and Brown and Kim et al.,Marine Biotechnology, Vol. 4 (2002), pp. 63-73). Chen reported that theplasmid pBinUΩNP-1, the ubi1 promoter, and the Agrobacterium tumefaciensnopaline synthase gene 3′UTR/terminator are suitable to enable exogenousgene expression in Chlorella ellipsoidea. In addition, Chen reportedthat the G418 resistance cassette encoded on pBinUΩNP-1 was suitable foruse as a selectable marker in Chlorella ellipsoidea.

In an embodiment of the present invention, vector pBinUΩNP-1, comprisingthe nucleotide sequence encoding the aph 3′ gene product, conferringresistance to G418, for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellaellipsoidea to reflect the codon bias inherent in nuclear genes ofChlorella ellipsoidea in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Zea mays ubi1promoter upstream of the protein-coding sequence and operably linked tothe Agrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator atthe 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella ellipsoidea genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella ellipsoidea with the transformationvector is achieved through well-known transformation techniquesincluding electroporation or other known methods. Activity of the aph 3′gene product can be used as a marker to select for Chlorella ellipsoideatransformed with the transformation vector on, but not limited to, Knopagar medium comprising G418. Growth media suitable for Chlorellaellipsoidea lipid production include, but are not limited to, Knopmedium and those culture medium reported by Jarvis and Brown and Kim etal. Evaluation of fatty acid profiles of Chlorella ellipsoidea lipidscan be assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 13 Engineering Chlorella kessleri

Expression of recombinant genes in accordance with the present inventionin Chlorella kessleri can be accomplished by modifying the methods andvectors taught by El-Sheekh et al. as discussed herein. Briefly,El-Sheekh et al., Biologia Plantarium, Vol. 42:2 (1999), pp. 209-216,reported the stable transformation of Chlorella kessleri with plasmidDNA. Using the transformation method of microprojectile bombardment,El-Sheekh introduced the plasmid pBI121 (GenBank Accession No. AF485783)into Chlorella kessleri. Plasmid pBI121 comprised a kanamycin/neomycinantibiotic resistance cassette. This kanamycin/neomycin antibioticresistance cassette comprised the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter, sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) for resistance to kanamycin and G418, and the 3′UTR/terminator of the Agrobacterium tumefaciens nopaline synthase (nos)gene. pBI121 further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably linked to a CaMV 35S promoter andoperably linked to a 3′ UTR/terminator of the nos gene. Prior totransformation, Chlorella kessleri was unable to be propagated inculture medium comprising 15 ug/L kanamycin. Upon transformation withthe pBI121plasmid, transformants of Chlorella kessleri were obtainedthat were propagated in selective culture medium comprising 15 mg/Lkanamycin. The express ion of the nptII gene product in Chlorellakessleri enabled propagation in the presence of 15 mg/L kanamycin,thereby establishing the utility of the kanamycin/neomycin antibioticresistance cassette as selectable marker for use in Chlorella kessleri.Detectable activity of the GUS gene product indicated that the CaMV 35Spromoter and nos 3′ UTR are suitable for enabling heterologous geneexpression in Chlorella kessleri. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. As reported byEl-Sheekh, selection and maintenance of transformed Chlorella kessleriwas conducted on semisolid agar plates comprising YEG medium (1% yeastextract, 1% glucose) and 15 mg/L kanamycin. El-Sheekh also reported thepropagation of Chlorella kessleri in YEG liquid culture media.Additional media suitable for culturing Chlorella kessleri for lipidproduction are disclosed in Sato et al., BBA Molecular and Cell Biologyof Lipids, Vol. 1633 (2003), pp. 27-34). El-Sheekh reported that theplasmid pBI121, the CaMV promoter, and the nopaline synthase gene3′UTR/terminator are suitable to enable heterologous gene expression inChlorella kessleri. In addition, El-Sheekh reported that thekanamycin/neomycin resistance cassette encoded on pBI121 was suitablefor use as a selectable marker in Chlorella kessleri.

In an embodiment of the present invention, vector pBI121, comprising thenucleotide sequence encoding the kanamycin/neomycin resistance geneproduct for use as a selectable marker, is constructed and modified tofurther comprise a lipid biosynthesis pathway expression cassettesequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellakessleri to reflect the codon bias inherent in nuclear genes ofChlorella kessleri in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the CaMV 35S promoterupstream of the protein-coding sequence and operably linked to theAgrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella kessleri genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella kessleri with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nptII gene product can be used as a marker to select forChlorella kessleri transformed with the transformation vector on, butnot limited to, YEG agar medium comprising kanamycin or neomycin. Growthmedia suitable for Chlorella kessleri lipid production include, but arenot limited to, YEG medium, and those culture media reported by Sato etal. Evaluation of fatty acid profiles of Chlorella kessleri lipids canbe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 14 Engineering Dunaliella tertiolecta

Expression of recombinant genes in accordance with the present inventionin Dunaliella tertiolecta can be accomplished by modifying the methodsand vectors taught by Walker et al. as discussed herein. Briefly, Walkeret al., Journal of Applied Phycology, Vol. 17 (2005), pp. 363-368,reported stable nuclear transformation of Dunaliella tertiolecta withplasmid DNA. Using the transformation method of electroporation, Walkerintroduced the plasmid pDbleFLAG1.2 into Dunaliella tertiolecta.pDbleFLAG1.2 comprised sequence encoding a bleomycin antibioticresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotic phleomycin, operably linked to the promoter and 3′ UTR of theDunaliella tertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenasesmall subunit gene (rbcS1, GenBank Accession No. AY530155). Prior totransformation, Dunaliella tertiolecta was unable to be propagated inculture medium comprising 1 mg/L phleomycin. Upon transformation withthe pDbleFLAG1.2 plasmid, transformants of Dunaliella tertiolecta wereobtained that were propagated in selective culture medium comprising 1mg/L phleomycin. The expression of the ble gene product in Dunaliellatertiolecta enabled propagation in the presence of 1 mg/L phleomycin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Dunaliella tertiolecta.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. As reported by Walker, selection and maintenanceof transformed Dunaliella tertiolecta was conducted in Dunaliella medium(DM, as described by Provasoli et al., Archiv fur Mikrobiologie, Vol. 25(1957), pp. 392-428) further comprising 4.5 g/L NaCl and 1 mg/Lpheomycin. Additional media suitable for culturing Dunaliellatertiolecta for lipid production are discussed in Takagi et al., Journalof Bioscience and Bioengineering, Vol. 101:3 (2006), pp. 223-226 and inMassart and Hanston, Proceedings Venice 2010, Third InternationalSymposium on Energy from Biomass and Waste. Walker reported that theplasmid pDbleFLAG1.2 and the promoter and 3′ UTR of the Dunaliellatertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenase smallsubunit gene are suitable to enable heterologous expression inDunaliella tertiolecta. In addition, Walker reported that the bleomycinresistance cassette encoded on pDbleFLAG1.2 was suitable for use as aselectable marker in Dunaliella tertiolecta.

In an embodiment of the present invention, vector pDbleFLAG1.2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Dunaliella tertiolecta to reflect thecodon bias inherent in nuclear genes of Dunaliella tertiolecta inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the rbcS1 promoter upstream of the protein-codingsequence and operably linked to the rbcS1 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella tertiolecta genome for targeted genomic integration ofthe transformation vector. Homology regions may be selected to disruptone or more genomic sites of endogenous lipid biosynthesis pathwaygenes. Stable transformation of Dunaliella tertiolecta with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product can be used as a marker to select for Dunaliellatertiolecta transformed with the transformation vector on, but notlimited to, DM medium comprising pheomycin. Growth medium suitable forDunaliella tertiolecta lipid production include, but are not limited toDM medium and those culture media described by Takagi et al. and Massartand Hanston. Evaluation of fatty acid profiles of Dunaliella tertiolectalipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 15 Engineering Volvox carteri

Expression of recombinant genes in accordance with the present inventionin Volvox carteri can be accomplished by modifying the methods andvectors taught by Hallman and Rappel et al. as discussed herein.Briefly, Hallman and Rappel et al., The Plant Journal, Volume 17 (1999),pp. 99-109, reported the stable nuclear transformation of Volvox carteriwith plasmid DNA. Using the transformation method of microprojectilebombardment, Hallman and Rappel introduced the pzeoE plasmid into Volvoxcarteri. The pzeoE plasmid comprised sequence encoding a bleomycinantibiotic resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein (ble), forresistance to the antibiotic zeocin, operably linked to and the promoterand 3′ UTR of the Volvox carteri beta-tubulin gene (GenBank AccessionNo. L24547). Prior to transformation, Volvox carteri was unable to bepropagated in culture medium comprising 1.5 ug/ml zeocin. Upontransformation with the pzeoE plasmid, transformants of Volvox carteriwere obtained that were propagated in selective culture mediumcomprising greater than 20 ug/ml zeocin. The expression of the ble geneproduct in Volvox carteri enabled propagation in the presence of 20ug/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use in Volvoxcarteri. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. As reported by Hallman and Rappel,selection and maintenance of transformed Volvox carteri was conducted inVolvox medium (VM, as described by Provasoli and Pintner, The Ecology ofAlgae, Special Publication No. 2 (1959), Tyron, C. A. and Hartman, R.T., eds., Pittsburgh: University of Pittsburgh, pp. 88-96) with 1 mg/Lpheomycin. Media suitable for culturing Volvox carteri for lipidproduction are also discussed by Starr in Starr R. C., Dev Biol Suppl.,Vol. 4 (1970), pp. 59-100). Hallman and Rappel reported that the plasmidpzeoE and the promoter and 3′ UTR of the Volvox carteri beta-tubulingene are suitable to enable heterologous expression in Volvox carteri.In addition, Hallman and Rappel reported that the bleomycin resistancecassette encoded on pzeoE was suitable for use as a selectable marker inVolvox carteri. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inVolvox carteri and suitable for use as selective markers Volvox carteriin have been reported (for instance see Hallamann and Sumper,Proceedings of the National Academy of Sciences, Vol. 91 (1994), pp11562-11566 and Hallman and Wodniok, Plant Cell Reports, Volume 25(2006), pp. 582-581).

In an embodiment of the present invention, vector pzeoE, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 19, each protein-coding sequencecodon-optimized for expression in Volvox carteri to reflect the codonbias inherent in nuclear genes of Volvox carteri in accordance withTables 19A-D. For each lipid biosynthesis pathway protein of Table 20,the codon-optimized gene sequence can individually be operably linked tothe Volvox carteri beta-tubulin promoter upstream of the protein-codingsequence and operably linked to the Volvox carteri beta-tubulin3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Volvox carteri genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Volvox carteri genome(referenced in the publication by Prochnik et al., Science, Vol.329:5988 (2010), pp 223-226). Stable transformation of Volvox carteriwith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the ble gene product can be used as a markerto select for Volvox carteri transformed with the transformation vectoron, but not limited to, VM medium comprising zeocin. Growth mediumsuitable for Volvox carteri lipid production include, but are notlimited to VM medium and those culture media discussed by Starr.Evaluation of fatty acid profiles of Volvox carteri lipids can beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 16 Engineering Haematococcus pluvialis

Expression of recombinant genes in accordance with the present inventionin Haematococcus pluvialis can be accomplished by modifying the methodsand vectors taught by Steinbrenner and Sandmann et al. as discussedherein. Briefly, Steinbrenner and Sandmann et al., Applied andEnvironmental Microbiology, Vol. 72:12 (2006), pp. 7477-7484, reportedthe stable nuclear transformation of Haematococcus pluvialis withplasmid DNA. Using the transformation method of microprojectilebombardment, Steinbrenner introduced the plasmid pPlat-pds-L504R intoHaematococcus pluvialis. The plasmid pPlat-pds-L504R comprised anorflurazon resistance cassette, which comprised the promoter,protein-coding sequence, and 3′UTR of the Haematococcus pluvialisphytoene desaturase gene (Pds, GenBank Accession No. AY781170), whereinthe protein-coding sequence of Pds was modified at position 504 (therebychanging a leucine to an arginine) to encode a gene product (Pds-L504R)that confers resistance to the herbicide norflurazon. Prior totransformation with pPlat-pds-L504R, Haematococcus pluvialis was unableto propagate on medium comprising 5 uM norflurazon. Upon transformationwith the pPlat-pds-L504R plasmid, transformants of Haematococcuspluvialis were obtained that were propagated in selective culture mediumcomprising 5 uM norflurazon. The expression of the Pds-L504R geneproduct in Haematococcus pluvialis enabled propagation in the presenceof 5 uM norflurazon, thereby establishing the utility of the norflurazonherbicide resistance cassette as selectable marker for use inHaematococcus pluvialis. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. As reported bySteinbrenner, selection and maintenance of transformed Haematococcuspluvialis was conducted on agar plates comprising OHA medium (OHM (0.41g/L KNO₃, 0.03 g/L Na₂HPO₄, 0.246 g/L MgSO₄.7H₂O, 0.11 g/L CaCl₂.2H₂O,2.62 mg/L Fe_((III))citrate×H₂O, 0.011 mg/L CoCl₂.6H₂O, 0.012 mg/LCuSO₄.5H₂O, 0.075 mg/L Cr₂O₃, 0.98 mg/L MnCl₂.4H₂O, 0.12 mg/LNa₂MoO₄×2H₂O, 0.005 mg/L SeO₂ and 25 mg/L biotin, 17.5 mg/L thiamine,and 15 mg/L vitamin B12), supplemented with 2.42 g/L Tris-acetate, and 5mM norflurazon. Propagation of Haematococcus pluvialis in liquid culturewas performed by Steinbrenner and Sandmann using basal medium (basalmedium as described by Kobayashi et al., Applied and EnvironmentalMicrobiology, Vol. 59 (1993), pp. 867-873). Steinbrenner and Sandmannreported that the pPlat-pds-L504R plasmid and promoter and 3′ UTR of theHaematococcus pluvialis phytoene desaturase gene are suitable to enableheterologous expression in Haematococcus pluvialis. In addition,Steinbrenner and Sandmann reported that the norflurazon resistancecassette encoded on pPlat-pds-L504R was suitable for use as a selectablemarker in Haematococcus pluvialis. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Haematococcus pluvialis have beenreported (see Kathiresan et al., Journal of Phycology, Vol. 45 (2009),pp 642-649).

In an embodiment of the present invention, vector pPlat-pds-L504R,comprising the nucleotide sequence encoding the Pds-L504R gene productfor use as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Haematococcus pluvialis to reflect thecodon bias inherent in nuclear genes of Haematococcus pluvialis inaccordance with Tables 19 A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Haematococcus pluvialis pds gene promoterupstream of the protein-coding sequence and operably linked to theHaematococcus pluvialis pds gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Haematococcuspluvialis genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Haematococcus pluvialis with the transformation vectoris achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thePds-L504R gene product can be used as a marker to select forHaematococcus pluvialis transformed with the transformation vector on,but not limited to, OHA medium comprising norflurazon. Growth mediasuitable for Haematococcus pluvialis lipid production include, but arenot limited to basal medium and those culture media described byKobayashi et al., Kathiresan et al, and Gong and Chen, Journal ofApplied Phycology, Vol. 9:5 (1997), pp. 437-444). Evaluation of fattyacid profiles of Haematococcus pluvialis lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 17 Engineering Closterium peracerosum-strigosum-littoralecomplex

Expression of recombinant genes in accordance with the present inventionin Closterium peracerosum-strigosum-littorale complex can beaccomplished by modifying the methods and vectors taught by Abe et al.as discussed herein. Briefly, Abe et al., Plant Cell Physiology, Vol.52:9 (2011), pp. 1676-1685, reported the stable nuclear transformationof Closterium peracerosum-strigosum-littorale complex with plasmid DNA.Using the transformation methods of microprojectile bombardment, Abeintroduced the plasmid pSA106 into Closteriumperacerosum-strigosum-littorale complex. Plasmid pSA106 comprised ableomycin resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein gene (ble,GenBank Accession No. CAA37050) operably linked to the promoter and 3′UTR of the Closterium peracerosum-strigosum-littorale complexChlorophyll a/b-binding protein gene (CAB, GenBank Accession No.AB363403). Prior to transformation with pSA106, Closteriumperacerosum-strigosum-littorale complex was unable to propagate onmedium comprising 3 ug/ml phleomycin. Upon transformation with pSA106,transformants of Closterium peracerosum-strigosum-littorale complex wereobtained that were propagated in selective culture medium comprising 3ug/ml phleomycin. The expression of the ble gene product in Closteriumperacerosum-strigosum-littorale complex enabled propagation in thepresence of 3 ug/ml phleomycin, thereby establishing the utility of thebleomycin antibiotic resistance cassette as selectable marker for use inClosterium peracerosum-strigosum-littorale complex. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. As reported by Abe, selection and maintenance of transformedClosterium peracerosum-strigosum-littorale complex was conducted firstin top agar with C medium (0.1 g/L KNO₃, 0.015 g/L Ca(NO₃)₂.4H₂O, 0.05g/L glycerophosphate-Na2, 0.04 g/L MgSO₄.7H₂O, 0.5 g/L Tris(hydroxylmethyl) aminomethane, trace minerals, biotin, vitamins B₁ andB₁₂) and then subsequently isolated to agar plates comprising C mediumsupplemented with phleomycin. As reported by Abe, propagation ofClosterium peracerosum-strigosum-littorale complex in liquid culture wasperformed in C medium. Additional liquid culture medium suitable forpropagation of Closterium peracerosum-strigosum-littorale complex arediscussed by Sekimoto et al., DNA Research, 10:4 (2003), pp. 147-153.Abe reported that the pSA106 plasmid and promoter and 3′ UTR of theClosterium peracerosum-strigosum-littorale complex CAB gene are suitableto enable heterologous gene expression in Closteriumperacerosum-strigosum-littorale complex. In addition, Abe reported thatthe bleomycin resistance cassette encoded on pSA106 was suitable for useas a selectable marker in Closterium peracerosum-strigosum-littoralecomplex. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inClosterium peracerosum-strigosum-littorale complex have been reported(see Abe et al., Plant Cell Physiology, Vol. 49 (2008), pp. 625-632).

In an embodiment of the present invention, vector pSA106, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Closteriumperacerosum-strigosum-littorale complex to reflect the codon biasinherent in nuclear genes of Closterium peracerosum-strigosum-littoralecomplex in accordance with Tables 19A-D. For each lipid biosynthesispathway protein of Table 20, the codon-optimized gene sequence canindividually be operably linked to the Closteriumperacerosum-strigosum-littorale complex CAB gene promoter upstream ofthe protein-coding sequence and operably linked to the Closteriumperacerosum-strigosum-littorale complex CAB gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Closterium peracerosum-strigosum-littorale complex genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofClosterium peracerosum-strigosum-littorale complex with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the ble gene product can be used as a marker to select forClosterium peracerosum-strigosum-littorale complex transformed with thetransformation vector on, but not limited to, C medium comprisingphleomycin. Growth media suitable for Closteriumperacerosum-strigosum-littorale complex lipid production include, butare not limited to C medium and those culture media reported by Abe etal. and Sekimoto et al. Evaluation of fatty acid profiles of Closteriumperacerosum-strigosum-littorale complex lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 18 Engineering Dunaliella viridis

Expression of recombinant genes in accordance with the present inventionin Dunaliella viridis can be accomplished by modifying the methods andvectors taught by Sun et al. as discussed herein. Briefly, Sun et al.,Gene, Vol. 377 (2006), pp. 140-149, reported the stable transformationof Dunaliella viridis with plasmid DNA. Using the transformation methodof electroporation, Sun introduced the plasmid pDVNR, encoding the fullDunaliella viridis nitrate reductase gene into mutant Dunaliella viridis(Dunaliella viridis NR-mutants.) The NR-mutants are incapable of growthwithout the use of nitrate as a source of nitrogen. Nitrate reductasecatalyzes the conversion of nitrate to nitrite. Prior to transformation,Dunaliella viridis NR-mutants were unable to propagate in culture mediumcomprising nitrate (NO₃ ⁻) as the sole nitrogen source. The expressionof the Dunaliella viridis NR gene product in NR-mutant Dunaliellaviridis was used as a selectable marker to rescue the nitrate metabolismdeficiency. Upon transformation with the pDVNR plasmid, NR-mutantDunaliella viridis stably expressing the Dunaliella viridis NR geneproduct were obtained that were able to grow on agar plates comprisingnitrate as the sole carbon source. Evaluation of the DNA of the stabletransformants was performed by Southern analysis. Selection andmaintenance of the transformed Dunaliella viridis (NR mutant) wasperformed on agar plates comprising 5 mM KNO₃. Sun also reported thepropagation of Dunaliella viridis and Dunaliella viridis NR mutants inliquid culture medium. Additional media suitable for propagation ofDunaliella viridis are reported by Gordillo et al., Journal of AppliedPhycology, Vol. 10:2 (1998), pp. 135-144 and by Moulton and Burford,Hydrobiologia, Vols. 204-205:1 (1990), pp. 401-408. Sun reported thatthe plasmid pDVNR and the promoter and 3′ UTR/terminator of theDunaliella viridis nitrate reductase gene were suitable to enableheterologous expression in Dunaliella viridis NR-mutants. Sun alsoreported that expression of the Dunaliella viridis nitrate reductasegene product was suitable for use as a selectable marker in Dunaliellaviridis NR-mutants.

In an embodiment of the present invention, vector pDVNR, comprising thenucleotide sequence encoding the Dunaliella viridis nitrate reductase(DvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected Table 20, each protein-codingsequence codon-optimized for expression in Dunaliella viridis to reflectthe codon bias inherent in nuclear genes of Dunaliella viridis inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the DvNR promoter upstream of the protein-codingsequence and operably linked to the DvNR 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella viridis genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Dunaliella viridis NR mutants with thetransformation vector is achieved through well-known transformationtechniques including electorporation or other known methods. Activity ofthe DvNR gene product can be used as a selectable marker to rescue thenitrogen assimilation deficiency of Dunaliella viridis NR mutant strainsand to select for Dunaliella viridis NR-mutants stably expressing thetransformation vector. Growth media suitable for Dunaliella viridislipid production include, but are not limited to those discussed by Sunet al., Moulton and Burford, and Gordillo et al. Evaluation of fattyacid profiles of Dunaliella viridis lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 19 Engineering Dunaliella salina

Expression of recombinant genes in accordance with the present inventionin Dunaliella salina can be accomplished by modifying the methods andvectors taught by Geng et al. as discussed herein. Briefly, Geng et al.,Journal of Applied Phycology, Vol. 15 (2003), pp. 451-456, reported thestable transformation of Dunaliella salina with plasmid DNA. Using thetransformation method of electroporation, Geng introduced thepUΩHBsAg-CAT plasmid into Dunaliella salina. pUΩHBsAg-CAT comprises ahepatitis B surface antigen (HBsAG) expression cassette comprisingsequence encoding the hepatitis B surface antigen operably linked to aZea mays ubi1 promoter upstream of the HBsAG protein-coding region andoperably linked to the 3′UTR/terminator of the Agrobacterium tumefaciensnopaline synthase gene (nos) downstream of the HBsAG protein-codingregion. pUΩHBsAg-CAT further comprised a chloramphenicol resistancecassette, comprising sequence encoding the chloramphenicolacetyltransferase (CAT) gene product, conferring resistance to theantibiotic chloramphenicol, operably linked to the simian virus 40promoter and enhancer. Prior to transformation with pUΩHBsAg-CAT,Dunaliella salina was unable to propagate on medium comprising 60 mg/Lchloramphenicol. Upon transformation with the pUΩHBsAg-CAT plasmid,transformants of Dunaliella salina were obtained that were propagated inselective culture medium comprising 60 mg/L chloramphenicol. Theexpression of the CAT gene product in Dunaliella salina enabledpropagation in the presence of 60 mg/L chloramphenicol, therebyestablishing the utility of the chloramphenicol resistance cassette asselectable marker for use in Dunaliella salina. Detectable activity ofthe HBsAg gene product indicated that ubi1 promoter and nos3′UTR/terminator are suitable for enabling gene expression in Dunaliellasalina. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. Geng reported that selection andmaintenance of the transformed Dunaliella salina was performed on agarplates comprising Johnson's medium (J1, described by Borowitzka andBorowitzka (eds), Micro-algal Biotechnology. Cambridge University Press,Cambridge, pp. 460-461) with 60 mg/L chloramphenicol. Liquid propagationof Dunaliella salina was performed by Geng in J1 medium with 60 mg/Lchloramphenicol. Propagation of Dunaliella salina in media other than J1medium has been discussed (see Feng et al., Mol. Bio. Reports, Vol. 36(2009), pp. 1433-1439 and Borowitzka et al., Hydrobiologia, Vols.116-117:1 (1984), pp. 115-121). Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Dunaliella salina have been reported byFeng et al. Geng reported that the plasmid pUΩHBsAg-CAT, the ubi1promoter, and the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator are suitable to enable exogenous gene expression inDunaliella salina. In addition, Geng reported that the CAT resistancecassette encoded on pUΩHBsAg-CAT was suitable for use as a selectablemarker in Dunaliella salina.

In an embodiment of the present invention, vector pUΩHBsAg-CAT,comprising the nucleotide sequence encoding the CAT gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Dunaliella salina to reflect the codon bias inherentin nuclear genes of Dunaliella salina in accordance with Tables 19A-D.For each lipid biosynthesis pathway protein of Table 20, thecodon-optimized gene sequence can individually be operably linked to theubi1 promoter upstream of the protein-coding sequence and operablylinked to the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Dunaliella salina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Dunaliella salinawith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the CAT gene product can be used as a selectablemarker to select for Dunaliella salina transformed with thetransformation vector in, but not limited to, J1 medium comprisingchloramphenicol. Growth medium suitable for Dunaliella salina lipidproduction include, but are not limited to J1 medium and those culturemedia described by Feng et al. and Borowitzka et al. Evaluation of fattyacid profiles of Dunaliella salina lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 20 Engineering Gonium pectoral

Expression of recombinant genes in accordance with the present inventionin Gonium pectoral can be accomplished by modifying the methods andvectors taught by Lerche and Hallman et al. as discussed herein.Briefly, Lerche and Hallman et al., BMC Biotechnology, Volume 9:64,2009, reported the stable nuclear transformation of Gonium pectoralewith plasmid DNA. Using the transformation method of microprojectilebombardment, Lerche introduced the plasmid pPmr3 into Gonium pectorale.Plasmid pPmr3 comprised a paromomycin resistance cassette, comprising asequence encoding the aminoglycoside 3′-phosphotransferase (aphVIII)gene product (GenBank Accession No. AAB03856) of Streptomyces rimosusfor resistance to the antibiotic paromomycin, operably linked to theVolvox carteri hsp70A-rbcS3 hybrid promoter upstream of the aphVIIIprotein-coding region and operably linked to the 3′ UTR/terminator ofthe Volvox carteri rbcS3 gene downstream of the aphVIII protein-codingregion. Prior to transformation with pPmr3, Gonium pectorale was unableto propagate on medium comprising 0.06 ug/ml paromomycin. Upontransformation with pPmr3, transformants of Gonium pectorale wereobtained that were propagated in selective culture medium comprising0.75 and greater ug/ml paromomycin. The expression of the aphVIII geneproduct in Gonium pectorale enabled propagation in the presence of 0.75and greater ug/ml paromomycin, thereby establishing the utility of theparomomycin antibiotic resistance cassette as selectable marker for usein Gonium pectorale. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Lerche and Hallmanreported that selection and maintenance of the transformed Goniumpectorale was performed in liquid Jaworski's medium (20 mg/LCa(NO₃)₂.4H₂O, 12.4 mg/L KH₂PO₄, 50 mg/L MgSO₄.7H₂O, 15.9 mg/L NaHCO₃,2.25 mg/L EDTA-FeNa, 2.25 mg/L EDTA Na₂, 2.48 g/L H₃BO₃, 1.39 g/LMnCl₂.4H₂O, 1 mg/L (NH₄)₆MO₇O₂4.4H₂O, 0.04 mg/L vitamin B12, 0.04 mg/LThiamine-HCl, 0.04 mg/L biotin, 80 mg/L NaNO₃, 36 mg/L Na₄HPO₄.12H₂O)with 1.0 ug/ml paromomycin. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Gonium pectorale are further discussedby Lerche and Hallman. Lerche and Hallman reported that the plasmidpPmr3, Volvox carteri hsp70A-rbcS3 hybrid promoter, and the 3′UTR/terminator of the Volvox carteri rbcS3 gene are suitable to enableexogenous gene expression in Gonium pectorale. In addition, Lerche andHallman reported that the paromomycin resistance cassette encoded pPmr3was suitable for use as a selectable marker in Gonium pectorale.

In an embodiment of the present invention, vector pPmr3, comprising thenucleotide sequence encoding the aphVIII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Gonium pectorale to reflect the codon bias inherent innuclear genes of Gonium pectorale in accordance with Tables 19A-D. Foreach lipid biosynthesis pathway protein of Table 20, the codon-optimizedgene sequence can individually be operably linked to the Volvox carterihsp70A-rbcS3 hybrid promoter upstream of the protein-coding sequence andoperably linked to the Volvox carteri rbcS3 gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Gonium pectorale genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Gonium pectorale with the transformation vectorcan be achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of theaphVIII gene product can be used as a selectable marker to select forGonium pectorale transformed with the transformation vector in, but notlimited to, Jaworski's medium comprising paromomycin. Growth mediasuitable for Gonium pectorale lipid production include Jaworski's mediumand media reported by Stein, American Journal of Botany, Vol. 45:9(1958), pp. 664-672. Evaluation of fatty acid profiles of Goniumpectorale lipids can be assessed through standard lipid extraction andanalytical methods described herein.

Example 21 Engineering Phaeodactylum tricornutum

Expression of recombinant genes in accordance with the present inventionin Phaeodactylum tricornutum can be accomplished by modifying themethods and vectors taught by Apt et al. as discussed herein. Briefly,Apt et al., Molecular and General Genetics, Vol. 252 (1996), pp.572-579, reported the stable nuclear transformation of Phaeodactylumtricornutum with vector DNA. Using the transformation technique ofmicroprojectile bombardment, Apt introduced the plasmid pfcpA intoPhaeodactylum tricornutum. Plasmid pfcpA comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics phleomycin and zeocin, operably linked to the promoter ofthe Phaeodactylum tricornutum fucoxanthin chlorophyll a binding proteingene (fcpA) upstream of the ble protein-coding region and operablylinked to the 3′ UTR/terminator of the Phaeodactylum tricornutum fcpAgene at the 3′ region, or downstream of the ble protein-coding region.Prior to transformation with pfcpA, Phaeodactylum tricornutum was unableto propagate on medium comprising 50 ug/ml zeocin. Upon transformationwith pfcpA, transformants of Phaeodactylum tricornutum were obtainedthat were propagated in selective culture medium comprising 50 ug/mlzeocin. The expression of the ble gene product in Phaeodactylumtricornutum enabled propagation in the presence of 50 ug/ml zeocin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Phaeodactylum tricornutum.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Apt reported that selection and maintenance of thetransformed Phaeodactylum tricornutum was performed on agar platescomprising LDM medium (as reported by Starr and Zeikus, Journal ofPhycology, Vol. 29, Supplement, (1993)) with 50 mg/L zeocin. Aptreported liquid propagation of Phaeodactylum tricornutum transformantsin LDM medium with 50 mg/L zeocin. Propagation of Phaeodactylumtricornutum in medium other than LDM medium has been discussed (byZaslayskaia et al., Science, Vol. 292 (2001), pp. 2073-2075, and byRadokovits et al., Metabolic Engineering, Vol. 13 (2011), pp. 89-95).Additional plasmids, promoters, 3′UTR/terminators, and selectablemarkers suitable for enabling heterologous gene expression inPhaeodactylum tricornutum have been reported in the same report by Aptet al., by Zaslayskaia et al., and by Radokovits et al.). Apt reportedthat the plasmid pfcpA, and the Phaeodactylum tricornutum fcpA promoterand 3′ UTR/terminator are suitable to enable exogenous gene expressionin Phaeodactylum tricornutum. In addition, Apt reported that thebleomycin resistance cassette encoded on pfcpA was suitable for use as aselectable marker in Phaeodactylum tricornutum.

In an embodiment of the present invention, vector pfcpA, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Phaeodactylum tricornutum to reflect the codon biasinherent in nuclear genes of Phaeodactylum tricornutum in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the Phaeodactylum tricornutum fcpA gene promoter upstream ofthe protein-coding sequence and operably linked to the Phaeodactylumtricornutum fcpA gene 3′UTR/terminator at the 3′ region, or downstream,of the protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Phaeodactylum tricornutumgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Phaeodactylumtricornutum genome (referenced in the publication by Bowler et al.,Nature, Vol. 456 (2008), pp. 239-244). Stable transformation ofPhaeodactylum tricornutum with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product canbe used as a marker to select for Phaeodactylum tricornutum transformedwith the transformation vector in, but not limited to, LDM mediumcomprising paromomycin. Growth medium suitable for Phaeodactylumtricornutum lipid production include, but are not limited to f/2 mediumas reported by Radokovits et al. Evaluation of fatty acid profiles ofPhaeodactylum tricornutum lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 22 Engineering Chaetoceros sp

Expression of recombinant genes in accordance with the present inventionin Chaetoceros sp. can be accomplished by modifying the methods andvectors taught by Yamaguchi et al. as discussed herein. Briefly,Yamaguchi et al., Phycological Research, Vol. 59:2 (2011), pp. 113-119,reported the stable nuclear transformation of Chaetoceros sp. withplasmid DNA. Using the transformation method of microprojectilebombardment, Yamaguchi introduced the plasmid pTpfcp/nat intoChaetoceros sp. pTpfcp/nat comprised a nourseothricin resistancecassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Chaetoceros sp. was unable to propagate on mediumcomprising 500 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Chaetoceros sp. were obtained that werepropagated in selective culture medium comprising 500 ug/mlnourseothricin. The expression of the nat gene product in Chaetocerossp. enabled propagation in the presence of 500 ug/ml nourseothricin,thereby establishing the utility of the nourseothricin antibioticresistance cassette as selectable marker for use in Chaetoceros sp.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Yamaguchi reported that selection and maintenanceof the transformed Chaetoceros sp. was performed on agar platescomprising f/2 medium (as reported by Guilard, R. R., Culture ofPhytoplankton for feeding marine invertebrates, In Culture of MarineInvertebrate Animals, Smith and Chanley (eds) 1975, Plenum Press, NewYork, pp. 26-60) with 500 ug/ml nourseothricin. Liquid propagation ofChaetoceros sp. transformants, as performed by Yamaguchi, was carriedout in f/2 medium with 500 mg/L nourseothricin. Propagation ofChaetoceros sp. in additional culture medium has been reported (forexample in Napolitano et al., Journal of the World Aquaculture Society,Vol. 21:2 (1990), pp. 122-130, and by Volkman et al., Journal ofExperimental Marine Biology and Ecology, Vol. 128:3 (1989), pp.219-240). Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inChaetoceros sp. have been reported in the same report by Yamaguchi etal. Yamaguchi reported that the plasmid pTpfcp/nat, and theThalassiosira pseudonana fcp promoter and 3′ UTR/terminator are suitableto enable exogenous gene expression in Chaetoceros sp. In addition,Yamaguchi reported that the nourseothricin resistance cassette encodedon pTpfcp/nat was suitable for use as a selectable marker in Chaetocerossp.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in the closely-related Chaetoceroscompressum to reflect the codon bias inherent in nuclear genes ofChaetoceros compressum in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Thalassiosirapseudonana fcp gene promoter upstream of the protein-coding sequence andoperably linked to the Thalassiosira pseudonana fcp gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chaetoceros sp. genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chaetoceros sp.with the transformation vector is achieved through well-knowntransformation including microprojectile bombardment or other knownmethods. Activity of the nat gene product can be used as a selectablemarker to select for Chaetoceros sp. transformed with the transformationvector in, but not limited to, f/2 agar medium comprisingnourseothricin. Growth medium suitable for Chaetoceros sp. lipidproduction include, but are not limited to, f/2 medium, and thoseculture media discussed by Napolitano et al. and Volkman et al.Evaluation of fatty acid profiles of Chaetoceros sp lipids can beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 23 Engineering Cylindrotheca fusiformis

Expression of recombinant genes in accordance with the present inventionin Cylindrotheca fusiformis can be accomplished by modifying the methodsand vectors taught by Poulsen and Kroger et al. as discussed herein.Briefly, Poulsen and Kroger et al., FEBS Journal, Vol. 272 (2005), pp.3413-3423, reported the transformation of Cylindrotheca fusiformis withplasmid DNA. Using the transformation method of microprojectilebombardment, Poulsen and Kroger introduced the pCF-ble plasmid intoCylindrotheca fusiformis. Plasmid pCF-ble comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics zeocin and phleomycin, operably linked to the Cylindrothecafusiformis fucozanthin chlorophyll a/c binding protein gene (fcpA,GenBank Accession No. AY125580) promoter upstream of the bleprotein-coding region and operably linked to the Cylindrothecafusiformis fcpA gene 3′UTR/terminator at the 3′ region (down-stream ofthe ble protein-coding region). Prior to transformation with pCF-ble,Cylindrotheca fusiformis was unable to propagate on medium comprising 1mg/ml zeocin. Upon transformation with pCF-ble, transformants ofCylindrotheca fusiformis were obtained that were propagated in selectiveculture medium comprising 1 mg/ml zeocin. The expression of the ble geneproduct in Cylindrotheca fusiformis enabled propagation in the presenceof 1 mg/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use inCylindrotheca fusiformis. Poulsen and Kroger reported that selection andmaintenance of the transformed Cylindrotheca fusiformis was performed onagar plates comprising artificial seawater medium with 1 mg/ml zeocin.Poulsen and Kroger reported liquid propagation of Cylindrothecafusiformis transformants in artificial seawater medium with 1 mg/mlzeocin. Propagation of Cylindrotheca fusiformis in additional culturemedium has been discussed (for example in Liang et al., Journal ofApplied Phycology, Vol. 17:1 (2005), pp. 61-65, and by Orcutt andPatterson, Lipids, Vol. 9:12 (1974), pp. 1000-1003). Additionalplasmids, promoters, and 3′UTR/terminators for enabling heterologousgene expression in Chaetoceros sp. have been reported in the same reportby Poulsen and Kroger. Poulsen and Kroger reported that the plasmidpCF-ble and the Cylindrotheca fusiformis fcp promoter and 3′UTR/terminator are suitable to enable exogenous gene expression inCylindrotheca fusiformis. In addition, Poulsen and Kroger reported thatthe bleomycin resistance cassette encoded on pCF-ble was suitable foruse as a selectable marker in Cylindrotheca fusiformis.

In an embodiment of the present invention, vector pCF-ble, comprisingthe nucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Cylindrotheca fusiformis to reflect the codon biasinherent in nuclear genes of Cylindrotheca fusiformis in accordance withTables 19A-D. For each lipid biosynthesis pathway protein of Table 20,the codon-optimized gene sequence can individually be operably linked tothe Cylindrotheca fusiformis fcp gene promoter upstream of theprotein-coding sequence and operably linked to the Cylindrothecafusiformis fcp gene 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Cylindrotheca fusiformisgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofCylindrotheca fusiformis with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product canbe used as a selectable marker to select for Cylindrotheca fusiformistransformed with the transformation vector in, but not limited to,artificial seawater agar medium comprising zeocin. Growth media suitablefor Cylindrotheca fusiformis lipid production include, but are notlimited to, artificial seawater and those media reported by Liang et al.and Orcutt and Patterson. Evaluation of fatty acid profiles ofCylindrotheca fusiformis lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 24 Engineering Amphidinium sp

Expression of recombinant genes in accordance with the present inventionin Amphidinium sp. can be accomplished by modifying the methods andvectors taught by ten Lohuis and Miller et al. as discussed herein.Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol. 13:3(1998), pp. 427-435, reported the stable transformation of Amphidiniumsp. with plasmid DNA. Using the transformation technique of agitation inthe presence of silicon carbide whiskers, ten Lohuis introduced theplasmid pMT NPT/GUS into Amphidinium sp. pMT NPT/GUS comprised aneomycin resistance cassette, comprising sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) operably linked to the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter upstream, or 5′ of the nptII protein-codingregion and operably linked to the 3′ UTR/terminator of the nos gene atthe 3′ region (down-stream of the nptII protein-coding region). ThenptII gene product confers resistance to the antibiotic G418. The pMTNPT/GUS plasmid further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably-linked to a CaMV 35S promoter andfurther operably linked to the CaMV 35S 3′ UTR/terminator. Prior totransformation with pMT NPT/GUS, Amphidinium sp. was unable to bepropagated on medium comprising 3 mg/ml G418. Upon transformation withpMT NPT/GUS, transformants of Amphidinium sp. were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Amphidinium sp. enabledpropagation in the presence of 3 mg/ml G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Amphidinium sp. Detectable activity of the GUSreporter gene indicated that CaMV 35S promoter and 3′UTR are suitablefor enabling gene expression in Amphidinium sp. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. ten Lohuis and Miller reported liquid propagation ofAmphidinium sp transformants in medium comprising seawater supplementedwith F/2 enrichment solution (provided by the supplier Sigma) and 3mg/ml G418 as well as selection and maintenance of Amphidinium sp.transformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Amphidinium sp. inadditional culture medium has been reported (for example in Mansour etal., Journal of Applied Phycology, Vol. 17:4 (2005) pp. 287-v300). Anadditional plasmid, comprising additional promoters, 3′UTR/terminators,and a selectable marker for enabling heterologous gene expression inAmphidinium sp. have been reported in the same report by ten Lohuis andMiller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUS andthe promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Amphidinium sp. Inaddition, ten Lohuis and Miller reported that the neomycin resistancecassette encoded on pMT NPT/GUS was suitable for use as a selectablemarker in Amphidinium sp.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Amphidinium sp. to reflect the codonbias inherent in nuclear genes of the closely-related species,Amphidinium carterae in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Agrobacteriumtumefaciens nopaline synthase (nos) gene promoter upstream of theprotein-coding sequence and operably linked to the nos 3′UTR/terminatorat the 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Amphidinium sp. genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Amphidinium sp. with the transformation vectoris achieved through well-known transformation techniques includingsilicon fibre-mediated microinjection or other known methods. Activityof the nptII gene product can be used as a selectable marker to selectfor Amphidinium sp. transformed with the transformation vector in, butnot limited to, seawater agar medium comprising G418. Growth mediasuitable for Amphidinium sp. lipid production include, but are notlimited to, artificial seawater and those media reported by Mansour etal. and ten Lohuis and Miller. Evaluation of fatty acid profiles ofAmphidinium sp. lipids can be assessed through standard lipid extractionand analytical methods described herein.

Example 25 Engineering Symbiodinium microadriacticum

Expression of recombinant genes in accordance with the present inventionin Symbiodinium microadriacticum can be accomplished by modifying themethods and vectors taught by ten Lohuis and Miller et al. as discussedherein. Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol.13:3 (1998), pp. 427-435, reported the stable transformation ofSymbiodinium microadriacticum with plasmid DNA. Using the transformationtechnique of silicon fibre-mediated microinjection, ten Lohuisintroduced the plasmid pMT NPT/GUS into Symbiodinium microadriacticum.pMT NPT/GUS comprised a neomycin resistance cassette, comprisingsequence encoding the neomycin phosphotransferase II (nptII) geneproduct (GenBank Accession No. AAL92039) operably linked to theAgrobacterium tumefaciens nopaline synthase (nos) gene promoterupstream, or 5′ of the nptII protein-coding region and operably linkedto the 3′ UTR/terminator of the nos gene at the 3′ region (down-streamof the nptII protein-coding region). The nptII gene product confersresistance to the antibiotic G418. The pMT NPT/GUS plasmid furthercomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter and further operablylinked to the CaMV 35S 3′ UTR/terminator. Prior to transformation withpMT NPT/GUS, Symbiodinium microadriacticum was unable to be propagatedon medium comprising 3 mg/ml G418. Upon transformation with pMT NPT/GUS,transformants of Symbiodinium microadriacticum were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Symbiodinium microadriacticumenabled propagation in the presence of 3 mg/ml G418, therebyestablishing the utility of the neomycin antibiotic resistance cassetteas selectable marker for use in Symbiodinium microadriacticum.Detectable activity of the GUS reporter gene indicated that CaMV 35Spromoter and 3′UTR are suitable for enabling gene expression inSymbiodinium microadriacticum. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. ten Lohuis andMiller reported liquid propagation of Symbiodinium microadriacticumtransformants in medium comprising seawater supplemented with F/2enrichment solution (provided by the supplier Sigma) and 3 mg/ml G418 aswell as selection and maintenance of Symbiodinium microadriacticumtransformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Symbiodiniummicroadriacticum in additional culture medium has been discussed (forexample in Iglesias-Prieto et al., Proceedings of the National Academyof Sciences, Vol. 89:21 (1992) pp. 10302-10305). An additional plasmid,comprising additional promoters, 3′UTR/terminators, and a selectablemarker for enabling heterologous gene expression in Symbiodiniummicroadriacticum have been discussed in the same report by ten Lohuisand Miller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUSand the promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Symbiodiniummicroadriacticum. In addition, ten Lohuis and Miller reported that theneomycin resistance cassette encoded on pMT NPT/GUS was suitable for useas a selectable marker in Symbiodinium microadriacticum.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Symbiodinium microadriacticum to reflect the codonbias inherent in nuclear genes of Symbiodinium microadriacticum inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Agrobacterium tumefaciens nopaline synthase(nos) gene promoter upstream of the protein-coding sequence and operablylinked to the nos 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Symbiodiniummicroadriacticum genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Symbiodinium microadriacticum with thetransformation vector is achieved through well-known transformationtechniques including silicon fibre-mediated microinjection or otherknown methods. Activity of the nptII gene product can be used as aselectable marker to select for Symbiodinium microadriacticumtransformed with the transformation vector in, but not limited to,seawater agar medium comprising G418. Growth media suitable forSymbiodinium microadriacticum lipid production include, but are notlimited to, artificial seawater and those media reported byIglesias-Prieto et al. and ten Lohuis and Miller. Evaluation of fattyacid profiles of Symbiodinium microadriacticum lipids can be assessedthrough standard lipid extraction and analytical methods describedherein.

Example 26 Engineering Nannochloropsis sp

Expression of recombinant genes in accordance with the present inventionin Nannochloropsis sp. W2J3B can be accomplished by modifying themethods and vectors taught by Kilian et al. as discussed herein.Briefly, Kilian et al., Proceedings of the National Academy of Sciences,Vol. 108:52 (2011) pp. 21265-21269, reported the stable nucleartransformation of Nannochloropsis with a transformation construct. Usingthe transformation method of electroporation, Kilian introduced thetransformation construct C2 into Nannochloropsis sp. W2J3B. The C2transformation construct comprised a bleomycin resistance cassette,comprising the coding sequence for the Streptoalloteichus hindustanusBleomycin binding protein (ble), for resistance to the antibioticsphleomycin and zeocin, operably linked to and the promoter of theNannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding proteingene VCP2 upstream of the ble protein-coding region and operably linkedto the 3′UTR/terminator of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding gene VCP1 downstream of the bleprotein-coding region. Prior to transformation with C2, Nannochloropsissp. W2J3B was unable to propagate on medium comprising 2 ug/ml zeocin.Upon transformation with C2, transformants of Nannochloropsis sp. W2J3Bwere obtained that were propagated in selective culture mediumcomprising 2 ug/ml zeocin. The expression of the ble gene product inNannochloropsis sp. W2J3B enabled propagation in the presence of 2 ug/mlzeocin, thereby establishing the utility of the bleomycin antibioticresistance cassette as selectable marker for use in Nannochloropsis.Evaluation of the genomic DNA of the stable transformants was performedby PCR. Kilian reported liquid propagation of Nannochloropsis sp. W2J3Btransformants in F/2 medium (reported by Guilard and Ryther, CanadianJournal of Microbiology, Vol. 8 (1962), pp. 229-239) comprising fivefoldlevels of trace metals, vitamins, and phosphate solution, and furthercomprising 2 ug/ml zeocin. Kilian also reported selection andmaintenance of Nannochloropsis sp. W2J3B transformants on agar F/2medium comprising artificial seawater 2 mg/ml zeocin. Propagation ofNannochloropsis in additional culture medium has been discussed (forexample in Chiu et al., Bioresour Technol., Vol. 100:2 (2009), pp.833-838 and Pal et al., Applied Microbiology and Biotechnology, Vol.90:4 (2011), pp. 1429-1441). Additional transformation constructs,comprising additional promoters and 3′UTR/terminators for enablingheterologous gene expression in Nannochloropsis sp. W2J3B and selectablemarkers for selection of transformants have been described in the samereport by Kilian. Kilian reported that the transformation construct C2and the promoter of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding protein gene VCP2 and 3′UTR/terminator of the Nannochloropsis sp. W2J3B violaxanthin/chlorophylla-binding protein gene VCP1 are suitable to enable exogenous geneexpression in Nannochloropsis sp. W2J3B. In addition, Kilian reportedthat the bleomycin resistance cassette encoded on C2 was suitable foruse as a selectable marker in Nannochloropsis sp. W2J3B.

In an embodiment of the present invention, transformation construct C2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Nannochloropsis sp. W2J3B to reflectthe codon bias inherent in nuclear genes of Nannochloropsis sp. inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Nannochloropsis sp. W2J3B VCP2 gene promoterupstream of the protein-coding sequence and operably linked to theNannochloropsis sp. W2J3B VCP1 gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to theNannochloropsis sp. W2J3B genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Nannochloropsis sp. W2J3B with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product can be used as a selectable marker to select forNannochloropsis sp. W2J3B transformed with the transformation vector in,but not limited to, F/2 medium comprising zeocin. Growth media suitablefor Nannochloropsis sp. W2J3B lipid production include, but are notlimited to, F/2 medium and those media reported by Chiu et al. and Palet al. Evaluation of fatty acid profiles of Nannochloropsis sp. W2J3Blipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 27 Engineering Cyclotella cryptica

Expression of recombinant genes in accordance with the present inventionin Cyclotella cryptica can be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Cyclotella cryptica with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Cyclotella cryptica. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Cyclotella cryptica was unable to propagate on 50%artificial seawater medium comprising 100 ug/ml G418. Upontransformation with pACCNPT5.1, transformants of Cyclotella crypticawere obtained that were propagated in selective 50% artificial seawatermedium comprising 100 ug/ml G418. The expression of the nptII geneproduct in Cyclotella cryptica enabled propagation in the presence of100 ug/ml G418, thereby establishing the utility of the neomycinantibiotic resistance cassette as selectable marker for use inCyclotella cryptica. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Dunahay reportedliquid propagation of Cyclotella cryptica in artificial seawater medium(ASW, as discussed by Brown, L., Phycologia, Vol. 21 (1982), pp.408-410) supplemented with 1.07 mM sodium silicate and with 100 ug/mlG418. Dunahay also reported selection and maintenance of Cyclotellacryptica transformants on agar plates comprising ASW medium with 100ug/ml G418. Propagation of Cyclotella cryptica in additional culturemedium has been discussed (for example in Sriharan et al., AppliedBiochemistry and Biotechnology, Vol. 28-29:1 (1991), pp. 317-326 andPahl et al., Journal of Bioscience and Bioengineering, Vol. 109:3(2010), pp. 235-239). Dunahay reported that the plasmid pACCNPT5.1 andthe promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase)gene are suitable to enable exogenous gene expression in Cyclotellacryptica. In addition, Dunahay reported that the neomycin resistancecassette encoded on pACCNPT5.1 was suitable for use as a selectablemarker in Cyclotella cryptica.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Cyclotella cryptica to reflect thecodon bias inherent in nuclear genes of Cyclotella cryptica inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Cyclotella cryptica ACCase promoter upstreamof the protein-coding sequence and operably linked to the Cyclotellacryptica ACCase 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Cyclotella cryptica genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Cyclotella crypticawith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the nptII gene product can be used as amarker to select for Cyclotella cryptica transformed with thetransformation vector in, but not limited to, agar ASW medium comprisingG418. Growth media suitable for Cyclotella cryptica lipid productioninclude, but are not limited to, ASW medium and those media reported bySriharan et al., 1991 and Pahl et al. Evaluation of fatty acid profilesof Cyclotella cryptica lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 28 Engineering Navicula saprophila

Expression of recombinant genes in accordance with the present inventionin Navicula saprophila can be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Navicula saprophila with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Navicula saprophila. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Navicula saprophila was unable to propagate on artificialseawater medium comprising 100 ug/ml G418. Upon transformation withpACCNPT5.1, transformants of Navicula saprophila were obtained that werepropagated in selective artificial seawater medium comprising 100 ug/mlG418. The expression of the nptII gene product in Navicula saprophilaenabled propagation in the presence of G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Navicula saprophila. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Dunahayreported liquid propagation of Navicula saprophila in artificialseawater medium (ASW, as discussed by Brown, L., Phycologia, Vol. 21(1982), pp. 408-410) supplemented with 1.07 mM sodium silicate and with100 ug/ml G418. Dunahay also reported selection and maintenance ofNavicula saprophila transformants on agar plates comprising ASW mediumwith 100 ug/ml G418. Propagation of Navicula saprophila in additionalculture medium has been discussed (for example in Tadros and Johansen,Journal of Phycology, Vol. 24:4 (1988), pp. 445-452 and Sriharan et al.,Applied Biochemistry and Biotechnology, Vol. 20-21:1 (1989), pp.281-291). Dunahay reported that the plasmid pACCNPT5.1 and the promoterof the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene aresuitable to enable exogenous gene expression in Navicula saprophila. Inaddition, Dunahay reported that the neomycin resistance cassette encodedon pACCNPT5.1 was suitable for use as a selectable marker in Naviculasaprophila.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Navicula saprophila to reflect thecodon bias inherent in nuclear genes of the closely-related Naviculapelliculosa in accordance with Tables 19A-D. For each lipid biosynthesispathway protein of Table 20, the codon-optimized gene sequence canindividually be operably linked to the Cyclotella cryptica ACCase genepromoter upstream of the protein-coding sequence and operably linked tothe Cyclotella cryptica ACCase gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to the Naviculasaprophila genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Navicula saprophila with the transformation vector isachieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thenptII gene product can be used as a selectable marker to select forNavicula saprophila transformed with the transformation vector in, butnot limited to, agar ASW medium comprising G418. Growth media suitablefor Navicula saprophila lipid production include, but are not limitedto, ASW medium and those media reported by Sriharan et al. 1989 andTadros and Johansen. Evaluation of fatty acid profiles of Naviculasaprophila lipids can be assessed through standard lipid extraction andanalytical methods described herein.

Example 29 Engineering Thalassiosira pseudonana

Expression of recombinant genes in accordance with the present inventionin Thalassiosira pseudonana can be accomplished by modifying the methodsand vectors taught by Poulsen et al. as discussed herein. Briefly,Poulsen et al., Journal of Phycology, Vol. 42 (2006), pp. 1059-1065,reported the stable transformation of Thalassiosira pseudonana withplasmid DNA. Using the transformation method of microprojectilebombardment, Poulsen introduced the plasmid pTpfcp/nat in toThalassiosira pseudonana. pTpfcp/nat comprised a nourseothricinresistance cassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Thalassiosira pseudonana was unable to propagate onmedium comprising 10 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Thalassiosira pseudonana were obtained thatwere propagated in selective culture medium comprising 100 ug/mlnourseothricin. The expression of the nat gene product in Thalassiosirapseudonana enabled propagation in the presence of 100 ug/mlnourseothricin, thereby establishing the utility of the nourseothricinantibiotic resistance cassette as selectable marker for use inThalassiosira pseudonana. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Poulsen reported thatselection and maintenance of the transformed Thalassiosira pseudonanawas performed in liquid culture comprising modified ESAW medium (asdiscussed by Harrison et al., Journal of Phycology, Vol. 16 (1980), pp.28-35) with 100 ug/ml nourseothricin. Propagation of Thalassiosirapseudonana in additional culture medium has been discussed (for examplein Volkman et al., Journal of Experimental Marine Biology and Ecology,Vol. 128:3 (1989), pp. 219-240). An additional plasmid, comprisingadditional selectable markers suitable for use in Thalassiosirapseudonana has been discussed in the same report by Poulsen. Poulsenreported that the plasmid pTpfcp/nat, and the Thalassiosira pseudonanafcp promoter and 3′ UTR/terminator are suitable to enable exogenous geneexpression in Thalassiosira pseudonana. In addition, Poulsen reportedthat the nourseothricin resistance cassette encoded on pTpfcp/nat wassuitable for use as a selectable marker in Thalassiosira pseudonana.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Thalassiosira pseudonana to reflectthe codon bias inherent in nuclear genes of Thalassiosira pseudonana inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Thalassiosira pseudonana fcp gene promoterupstream of the protein-coding sequence and operably linked to theThalassiosira pseudonana fcp gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Thalassiosirapseudonana genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. One skilled in theart can identify such homology regions within the sequence of theThalassiosira pseudonana genome (referenced in the publication byArmbrust et al., Science, Vol. 306: 5693 (2004): pp. 79-86). Stabletransformation of Thalassiosira pseudonana with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nat gene product can be used as a marker to select forThalassiosira pseudonana transformed with the transformation vector inbut not limited to, ESAW agar medium comprising nourseothricin. Growthmedia suitable for Thalassiosira pseudonana lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Volkman et al. and Harrison et al. Evaluation of fatty acid profilesof Thalassiosira pseudonana lipids can be assessed through standardlipid extraction and analytical methods described herein.

Example 30 Engineering Chlamydomonas reinhardtii

Expression of recombinant genes in accordance with the present inventionin Chlamydomonas reinhardtii can be accomplished by modifying themethods and vectors taught by Cerutti et al. as discussed herein.Briefly, Cerutti et al., Genetics, Vol. 145:1 (1997), pp. 97-110,reported the stable nuclear transformation of Chlamydomonas reinhardtiiwith a transformation vector. Using the transformation method ofmicroprojectile bombardment, Cerutti introduced transformation constructP[1030] into Chlamydomonas reinhardtii. Construct P[1030] comprised aspectinomycin resistance cassette, comprising sequence encoding theaminoglucoside 3″-adenyltransferase (aadA) gene product operably linkedto the Chlamydomonas reinhardtii ribulose-1,5-bisphosphatecarboxylase/oxygenase small subunit gene (RbcS2, GenBank Accession No.X04472) promoter upstream of the aadA protein-coding region and operablylinked to the Chlamydomonas reinhardtii RbcS2 gene 3′ UTR/terminator atthe 3′ region (downstream of the aadA protein coding-sequence). The aadAgene product confers resistance to the antibiotic spectinomycin. Priorto transformation with P[1030], Chlamydomonas reinhardtii was unable topropagate on medium comprising 90 ug/ml spectinomycin. Upontransformation with P[1030], transformants of Chlamydomonas reinhardtiiwere obtained that were propagated in selective culture mediumcomprising 90 ug/ml spectinomycin, thereby establishing the utility ofthe spectinomycin antibiotic resistance cassette as a selectable markerfor use in Chlamydomonas reinhardtii. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Ceruttireported that selection and maintenance of the transformed Chlamydomonasreinhardtii was performed on agar plates comprisingTris-acetate-phosphate medium (TAP, as described by Harris, TheChlamydomonas Sourcebook, Academic Press, San Diego, 1989) with 90 ug/mlspectinomycin. Cerutti additionally reported propagation ofChlamydomonas reinhardtii in TAP liquid culture with 90 ug/mlspectinomycin. Propagation of Chlamydomonas reinhardtii in alternativeculture medium has been discussed (for example in Dent et al., AfricanJournal of Microbiology Research, Vol. 5:3 (2011), pp. 260-270 andYantao et al., Biotechnology and Bioengineering, Vol. 107:2 (2010), pp.258-268). Additional constructs, comprising additional selectablemarkers suitable for use in Chlamydomonas reinhardtii as well asnumerous regulatory sequences, including promoters and 3′ UTRs suitablefor promoting heterologous gene expression in Chlamydomonas reinhardtiiare known in the art and have been discussed (for a review, seeRadakovits et al., Eukaryotic Cell, Vol. 9:4 (2010), pp. 486-501).Cerutti reported that the transformation vector P[1030] and theChlamydomonas reinhardtii promoter and 3′ UTR/terminator are suitable toenable exogenous gene expression in Chlamydomonas reinhardtii. Inaddition, Cerutti reported that the spectinomycin resistance cassetteencoded on P[1030] was suitable for use as a selectable marker inChlamydomonas reinhardtii.

In an embodiment of the present invention, vector P[1030], comprisingthe nucleotide sequence encoding the aadA gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Chlamydomonas reinhardtii to reflectthe codon bias inherent in nuclear genes of Chlamydomonas reinhardtii inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Chlamydomonas reinhardtii RbcS2 promoterupstream of the protein-coding sequence and operably linked to theChlamydomonas reinhardtii RbcS2 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Chlamydomonasreinhardtii genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic site of an endogenous lipid biosynthesis pathway gene.One skilled in the art can identify such homology regions within thesequence of the Chlamydomonas reinhardtii genome (referenced in thepublication by Merchant et al., Science, Vol. 318:5848 (2007), pp.245-250). Stable transformation of Chlamydomonas reinhardtii with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the aadA gene product can be used as a marker to select forChlamydomonas reinhardtii transformed with the transformation vector on,but not limited to, TAP agar medium comprising spectinomycin. Growthmedia suitable for Chlamydomonas reinhardtii lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Yantao et al. and Dent et al. Evaluation of fatty acid profiles ofChlamydomonas reinhardtii lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 31 Engineering Yarrowia lipolytica

Expression of recombinant genes in accordance with the present inventionin Yarrowia lipolytica can be accomplished by modifying the methods andvectors taught by Fickers et al. as discussed herein. Briefly, Fickerset al., Journal of Microbiological Methods, Vol. 55 (2003), pp. 727-737,reported the stable nuclear transformation of Yarrowia lipolytica withplasmid DNA. Using a lithium acetate transformation method, Fickersintroduced the plasmid JMP123 into Yarrowia lipolytica. Plasmid JMP123comprised a hygromycin B resistance cassette, comprising sequenceencoding the hygromycin B phosphotransferase gene product (hph),operably-linked to the Yarrowia lipolytica LIP2 gene promoter (GenBankAccession No. AJ012632) upstream of the hph protein-coding region andoperably linked to the Yarrowia lipolytica LIP2 gene 3′UTR/terminatordownstream of the hph protein-coding region. Prior to transformationwith JMP123, Yarrowia lipolytica were unable to propagate on mediumcomprising 100 ug/ml hygromycin. Upon transformation with JMP123,transformed Yarrowia lipolytica were obtained that were able topropagate on medium comprising 100 ug/ml hygromycin, therebyestablishing the hygromycin B antibiotic resistance cassette as aselectable marker for use in Yarrowia lipolytica. The nucleotidesequence provided on JMP123 of the promoter and 3′UTR/terminator of theYarrowia lipolytica LIP2 gene served as donor sequences for homologousrecombination of the hph coding sequence into the LIP2 locus. Evaluationof the genomic DNA of the stable transformants was performed bySouthern. Fickers reported that selection and maintenance of thetransformed Yarrowia lipolytica was performed on agar plates comprisingstandard YPD medium (Yeast Extract Peptone Dextrose) with 100 ug/mlhygromycin. Liquid culturing of transformed Yarrowia lipolytica wasperformed in YPD medium with hygromycin. Other media and techniques usedfor culturing Yarrowia lipolytica have been reported and numerous otherplasmids, promoters, 3′ UTRs, and selectable markers for use in Yarrowialipolytica have been reported (for example see Pignede et al., Appliedand Environmental Biology, Vol. 66:8 (2000), pp. 3283-3289, Chuang etal., New Biotechnology, Vol. 27:4 (2010), pp. 277-282, and Barth andGaillardin, (1996), In: K, W. (Ed.), Nonconventional Yeasts inBiotechnology. Sprinter-Verlag, Berlin-Heidelber, pp. 313-388). Fickersreported that the transformation vector JMP123 and the Yarrowialipolytica LIP2 gene promoter and 3′ UTR/terminator are suitable toenable heterologous gene expression in Yarrowia lipolytica. In addition,Fickers reported that the hygromycin resistance cassette encoded onJMP123 was suitable for use as a selectable marker in Yarrowialipolytica.

In an embodiment of the present invention, vector JMP123, comprising thenucleotide sequence encoding the hph gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Yarrowia lipolytica to reflect thecodon bias inherent in nuclear genes of Yarrowia lipolytica inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Yarrowia lipolytica LIP2 gene promoterupstream of the protein-coding sequence and operably linked to theYarrowia lipolytica LIP2 gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Yarrowia lipolyticagenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Yarrowialipolytica genome (referenced in the publication by Dujun et al.,Nature, Vol. 430 (2004), pp. 35-44). Stable transformation of Yarrowialipolytica with the transformation vector is achieved through well-knowntransformation techniques including lithium acetate transformation orother known methods. Activity of the hph gene product can be used as amarker to select for Yarrowia lipolytica transformed with thetransformation vector on, but not limited to, YPD medium comprisinghygromycin. Growth media suitable for Yarrowia lipolytica lipidproduction include, but are not limited to, YPD medium, and thoseculture media described by Chuang et al. Evaluation of fatty acidprofiles of Yarrowia lipolytica lipids can be assessed through standardlipid extraction and analytical methods described herein.

Example 32 Engineering Mortierella alpine

Expression of recombinant genes in accordance with the present inventionin Mortierella alpine can be accomplished by modifying the methods andvectors taught by Mackenzie et al. as discussed herein. Briefly,Mackenzie et al., Applied and Environmental Microbiology, Vol. 66(2000), pp. 4655-4661, reported the stable nuclear transformation ofMortierella alpina with plasmid DNA. Using a protoplast transformationmethod, MacKenzie introduced the plasmid pD4 into Mortierella alpina.Plasmid pD4 comprised a hygromycin B resistance cassette, comprisingsequence encoding the hygromycin B phosphotransferase gene product(hpt), operably-linked to the Mortierella alpina histone H4.1 genepromoter (GenBank Accession No. AJ249812) upstream of the hptprotein-coding region and operably linked to the Aspergillus nidulansN-(5′-phophoribosyl)anthranilate isomerase (trpC) gene 3′UTR/terminatordownstream of the hpt protein-coding region. Prior to transformationwith pD4, Mortierella alpina were unable to propagate on mediumcomprising 300 ug/ml hygromycin. Upon transformation with pD4,transformed Mortierella alpina were obtained that were propagated onmedium comprising 300 ug/ml hygromycin, thereby establishing thehygromycin B antibiotic resistance cassette as a selectable marker foruse in Mortierella alpina. Evaluation of the genomic DNA of the stabletransformants was performed by Southern. Mackenzie reported thatselection and maintenance of the transformed Mortierella alpina wasperformed on PDA (potato dextrose agar) medium comprising hygromycin.Liquid culturing of transformed Mortierella alpina by Mackenzie wasperformed in PDA medium or in S2GYE medium (comprising 5% glucose, 0.5%yeast extract, 0.18% NH₄SO₄, 0.02% MgSO₄-7H₂O, 0.0001% FeCl₃-6H₂O, 0.1%,trace elements, 10 mM K₂HPO₄—NaH₂PO₄), with hygromycin. Other media andtechniques used for culturing Mortierella alpina have been reported andother plasmids, promoters, 3′ UTRs, and selectable markers for use inMortierella alpina have been reported (for example see Ando et al.,Applied and Environmental Biology, Vol. 75:17 (2009) pp. 5529-35 and Luet al., Applied Biochemistry and Biotechnology, Vol. 164:7 (2001), pp.979-90). Mackenzie reported that the transformation vector pD4 and theMortierella alpina histone H4.1 promoter and A. nidulans trpC gene 3′UTR/terminator are suitable to enable heterologous gene expression inMortierella alpina. In addition, Mackenzie reported that the hygromycinresistance cassette encoded on pD4 was suitable for use as a selectablemarker in Mortierella alpina.

In an embodiment of the present invention, vector pD4, comprising thenucleotide sequence encoding the hpt gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Mortierella alpina to reflect thecodon bias inherent in nuclear genes of Mortierella alpina in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the Mortierella alpina histone H4.1 gene promoter upstream ofthe protein-coding sequence and operably linked to the A. nidulans trpC3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Mortierella alpina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Mortierella alpina genome(referenced in the publication by Wang et al., PLOS One, Vol. 6:12(2011)). Stable transformation of Mortierella alpina with thetransformation vector is achieved through well-known transformationtechniques including protoplast transformation or other known methods.Activity of the hpt gene product can be used as a marker to select forMortierella alpina transformed with the transformation vector on, butnot limited to, PDA medium comprising hygromycin. Growth media suitablefor Mortierella alpina lipid production include, but are not limited to,S2GYE medium, and those culture media described by Lu et al. and Ando etal. Evaluation of fatty acid profiles of Mortierella alpina lipids canbe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 33 Engineering Rhodococcus opacus PD630

Expression of recombinant genes in accordance with the present inventionin Rhodococcus opacus PD630 can be accomplished by modifying the methodsand vectors taught by Kalscheuer et al. as discussed herein. Briefly,Kalscheuer et al., Applied and Environmental Microbiology, Vol. 52(1999), pp. 508-515, reported the stable transformation of Rhodococcusopacus with plasmid DNA. Using the transformation method ofelectroporation, Kalscheuer introduced the plasmid pNC9501 intoRhodococcus opacus PD630. Plasmid pNC9501 comprised a thiostreptonresistance (thior) cassette, comprising the full nucleotide sequence ofthe Streptomyces azureus 23S rRNA A1067 methyltransferase gene,including the gene's promoter and 3′ terminator sequence. Prior totransformation with pNC9501, Rhodococcus opacus was unable to propagateon medium comprising 1 mg/ml thiostrepton. Upon transformation ofRhodococcus opacus PD630 with pNC9501, transformants were obtained thatpropagated on culture medium comprising 1 mg/ml thiostrepton, therebyestablishing the use of the thiostrepton resistance cassette as aselectable marker in Rhodococcus opacus PD630. A second plasmiddescribed by Kalscheuer, pAK68, comprised the resistance thior cassetteas well as the gene sequences of the Ralstonia eutrophabeta-ketothiolase (phaB), acetoacetyl-CoA reductase (phaA), andpoly3-hydroxyalkanoic acid synthase (phaC) genes forpolyhydroxyalkanoate biosynthesis, driven by the lacZ promoter. UponpAK68 transformation of a Rhodococcus opacus PD630 strain deficient inpolyhydroxyalkanoate biosynthesis, transformed Rhodococcus opacus PD630were obtained that produced higher amounts of polyhydroxyalkanoates thanthe untransformed strain. Detectable activity of the introducedRalstonia eutropha phaB, phaA, and phaC enzymes indicted that theregulatory elements encoded on the pAK68 plasmid were suitable forheterologous gene expression in Rhodococcus opacus PD630. Kalscheuerreported that selection and maintenance of the transformed Rhodococcusopacus PD630 was performed on standard Luria Broth (LB) medium, nutrientbroth (NB), or mineral salts medium (MSM) comprising thiostrepton. Othermedia and techniques used for culturing Rhodococcus opacus PD630 havebeen described (for example see Kurosawa et al., Journal ofBiotechnology, Vol. 147:3-4 (2010), pp. 212-218 and Alverez et al.,Applied Microbial and Biotechnology, Vol. 54:2 (2000), pp. 218-223).Kalscheuer reported that the transformation vectors pNC9501 and pAK68,the promoters of the Streptomyces azureus 23S rRNA A1067methyltransferase gene and lacZ gene are suitable to enable heterologousgene expression in Rhodococcus opacus PD630. In addition, Kalscheuerreported that the thior cassette encoded on pNC9501 and pAK68 wassuitable for use as a selectable marker in Rhodococcus opacus PD630.

In an embodiment of the present invention, vector pNC9501, comprisingthe nucleotide sequence encoding the thior gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Rhodococcus opacus PD630 to reflectthe codon bias inherent in nuclear genes of Rhodococcus opacus inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the lacZ gene promoter upstream of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Rhodococcus opacus PD630 genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Rhodococcusopacus PD630 genome (referenced in the publication by Holder et al.,PLOS Genetics, Vol. 7:9 (2011). Transformation of Rhodococcus opacusPD630 with the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the Streptomyces azureus 23S rRNA A1067methyltransferase gene product can be used as a marker to select forRhodococcus opacus PD630 transformed with the transformation vector on,but not limited to, LB medium comprising thiostrepton. Growth mediasuitable Rhodococcus opacus PD630 lipid production include, but are notlimited to those culture media discussed by Kurosawa et al. and Alvarezet al. Evaluation of fatty acid profiles of Rhodococcus opacus PD630lipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 34 Engineering Microalgae for Fatty Acid Auxotrophy

Strain B of Example 3, Prototheca moriformis (UTEX 1435) engineered toexpress a Cuphea wrightii thioesterase (CwTE2), was used as the hostorganism for further genetic modification to knockout both endogenousthioesterase alleles, FATA1-1 and FATA1-2. Here, a first transformationconstruct was generated to integrate a neomycin expression cassette intoStrain B at the FATA1-1 locus. This construct, pSZ2226, included 5′ (SEQID NO: 30) and 3′ (SEQ ID NO: 31) homologous recombination targetingsequences (flanking the construct) to the FATA1-1 locus of the nucleargenome and a neomycin resistance protein-coding sequence under thecontrol of the C. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5)and the Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). ThisNeoR expression cassette is listed as SEQ ID NO: 15 and served as aselectable marker.

Upon transformation of pSZ2226 into Strain B, individual transformantswere selected on agar plates comprising sucrose and G418. A singleisolate, Strain H, was selected for further genetic modification. Asecond transformation construct, pSZ2236, was generated to integratepolynucleotides enabling expression of a thiamine selectable marker intoStrain H at the FATA1-2 locus. pSZ2236 included 5′ (SEQ ID NO: 32) and3′ (SEQ ID NO: 33) homologous recombination targeting sequences(flanking the construct) to the FATA1-2 genomic region for integrationinto the P. moriformis (UTEX 1435) nuclear genome and an A. thalianaTHIC protein coding region under the control of the C. protothecoidesactin promoter/5′UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This AtTHIC expression cassette is listed as SEQID NO: 23 and served as a selectable marker. Upon transformation ofStrain H with pSZ2236 to generate Strain I, individual transformants,were selected on agar plates comprising free fatty acids. Strain I wasable to propagate on agar plates and in medium lacking thiamine andsupplemented with free fatty acids.

Example 35 Engineering Microorganisms for Increased Production ofStearic Acid

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed with the plasmid construct pSZ2281 accordingto biolistic transformation methods as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. pSZ2281 included polynucleotides encoding RNAhairpins (SAD2hpC, SEQ ID NO: 34) to down-regulate the expression ofstearoyl-ACP desaturase, 5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2)homologous recombination targeting sequences (flanking the construct) tothe 6S genomic region for integration into the nuclear genome, and a S.cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), toexpress the protein sequence given in SEQ ID NO: 3, under the control ofC. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselectable marker. The polynucleotide sequence encoding the SAD2hpC RNAhairpin was under the control of the C. protothecoides actinpromoter/5′UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6).

Upon transformation of Strain J with construct pSZ2281, therebygenerating Strain K, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using standard fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX Strain J propagated on glucoseas a sole carbon source and three representative isolates of Strain K,propagated on sucrose as a sole carbon source, are presented in Table21.

TABLE 21 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cellsengineered to express a hairpin RNA construct targeting stearoyl ACPdesaturase gene/gene products. Area % Fatty acid Strain J Strain K-1Strain K-2 Strain K-3 Strain K-4 C8:0 0.02 C10:0 0.01 0.00 0.02 0.020.04 C12:0 0.03 0.05 0.05 0.05 0.08 C14:0 1.22 0.89 0.87 0.77 1.2 C16:026.75 29.23 28.96 27.55 28.06 C18:0 3.06 37.39 36.76 36.41 40.82 C18:159.62 23.90 24.76 26.92 22.02 C18:2 7.33 5.44 5.54 5.54 4.53 C18:3 0.14C20:0 1.43

The data presented in Table 21 show a clear impact of the expression ofSAD2 hairpin RNA construct on the C18:0 and C18:1 fatty acid profiles ofthe transformed organism. The fatty acid profiles of Strain Ktransformants comprising a SAD2 hairpin RNA construct demonstrated anincrease in the percentage of saturated C18:0 fatty acids with aconcomitant diminution of unsaturated C18:1 fatty acids. Fatty acidprofiles of the untransformed strain comprise about 3% C18:0. Fatty acidprofiles of the transformed strains comprise about 37% C18:0. These dataillustrate the successful expression and use of polynucleotides enablingexpression of a SAD RNA hairpin construct in Prototheca moriformis toalter the percentage of saturated fatty acids in the engineered hostmicrobes, and in particular in increasing the concentration of C18:0fatty acids and decreasing C18:1 fatty acids in microbial cells.

Also shown in Table 21, strain K4 had a yet further elevated level ofstearate. Strain K4 was created by inserting the construct of strainsK1-K3 into the SAD2B locus. Thus, by knocking out one copy of the SADgene and inhibiting the remaining copies at the RNA level, a furtherreduction in oleic acid and corresponding increase in stearate wasobtained. Triglyceride analysis of RBD oil obtained from strain K4showed about 12% POP, 27% POS and 18% SOS.

Example 36 Engineering Microorganisms for Increased Production of OleicAcid Through Knockdown of an Endogenous Acyl-ACP Thioesterase

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed independently with each of the constructspSZ2402-pSZ2407 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructspSZ2402-pSZ2407 included different polynucleotides encoding a hairpinRNA targeted against Prototheca moriformis FATA1 mRNA transcripts todown-regulate the expression of fatty acyl-ACP thioesterase, 5′ (SEQ IDNO: 1) and 3′ (SEQ ID NO: 2) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the nuclear genome, and a S. cerevisiae suc2 sucroseinvertase coding region (SEQ ID NO: 4) to express the protein sequencegiven in SEQ ID NO: 3 under the control of C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette islisted as SEQ ID NO: 7 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to each hairpinare listed in Table 22. The polynucleotide sequence encoding each RNAhairpin was under the control of the C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6).

TABLE 22 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain J. Plasmid construct Hairpin designation SEQ ID NO:pSZ2402 PmFATA-hpB SEQ ID NO: 40 pSZ2403 PmFATA-hpC SEQ ID NO: 41pSZ2404 PmFATA-hpD SEQ ID NO: 42 pSZ2405 PmFATA-hpE SEQ ID NO: 43pSZ2406 PmFATA-hpF SEQ ID NO: 44 pSZ2407 PmFATA-hpG SEQ ID NO: 45

Upon independent transformation of Strain J with each of the constructslisted in Table 22, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using standard fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis (UTEX 1435) Strain J propagated onglucose as a sole carbon source and representative isolates of eachtransformation of Strain J, propagated on sucrose as a sole carbonsource, are presented in Table 23.

TABLE 23 Fatty acid profiles of Prototheca moriformis (UTEX 1435)cellsengineered to express hairpin RNA constructs targeting fatty acyl-ACPthioesterase gene/gene products. Area % Fatty Acid Construct C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 Strain J untransformed 0 0.05 1.32 26.663.1 59.07 7.39 PmFATA-hpB 0.04 0.07 1.36 24.88 2.24 61.92 6.84 0 0.081.33 25.34 2.39 61.72 6.5 0 0.07 1.29 25.44 2.26 61.7 6.69 0 0.06 1.3325.1 2.37 61.56 6.87 PmFATA-hpC 0 0.08 1.18 22.03 1.71 63.8 8.63 0 0.071.21 24.5 2.23 62.32 7.19 0 0.08 1.29 24.93 2.24 62.02 7.01 0.05 0.061.29 25.45 2.26 61.81 6.76 PmFATA-hpD 0 0.02 0.68 15.8 1.88 72.64 6.96 00.03 0.78 17.56 1.7 71.8 6.03 0 0.03 0.92 19.04 2.03 68.82 7.05 0 0.041.27 23.14 2.25 65.27 6.07 PmFATA-hpE 0 0.03 0.79 18.55 2.13 69.66 6.770 0.04 1.11 21.01 1.74 65.18 8.55 0 0.03 1.08 21.11 1.54 64.76 8.87 00.03 1.17 21.93 1.71 63.89 8.77 PmFATA-hpF 0.03 0.04 0.34 8.6 1.69 78.088.87 0 0.03 0.49 10.2 1.52 76.97 8.78 0 0.03 1 20.47 2.22 66.34 7.45 00.03 1.03 21.61 1.88 65.39 7.76 PmFATA-hpG 0 0.03 1.03 20.57 2.36 64.738.75 0 0.03 1.2 24.39 2.47 61.9 7.49 0 0.04 1.29 24.14 2.29 61.41 8.22

The data presented in Table 23 show a clear impact of the expression ofFATA hairpin RNA constructs on the C18:0 and C18:1 fatty acid profilesof the transformed organism. The fatty acid profiles of Strain Jtransformants comprising a FATA hairpin RNA construct demonstrated anincrease in the percentage of C18:1 fatty acids with a concomitantdiminution of C16:0 and C18:0 fatty acids. Fatty acid profiles of theuntransformed Strain J are about 26.66% C16:0, 3% C18:0, and about 59%C18:1 fatty acids. In contrast, the fatty acid profiles of thetransformed strains comprise as low as 8.6% C16:0 and 1.54% C18:0 andgreater than 78% C18:1 fatty acids.

These data illustrate the utility and successful use of polynucleotideFATA RNA hairpin constructs in Prototheca moriformis to alter the fattyacids profile of engineered microbes, and in particular in increasingthe concentration of C18:1 fatty acids and decreasing C18:0 and C16:0fatty acids in microbial cells.

Example 37 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI or KASIV Overexpression

This example describes the use of recombinant polynucleotides thatencode KASI or KASIV enzymes to engineer microorganisms in which thefatty acid profiles of the transformed microorganisms have been enrichedin lauric acid, C10:0, and total saturated fatty acids.

Each of the constructs pSZD1132, pSZD1133, pSZD1134, or pSZD1201 wasused independently to transform Strain B of Example 3, Protothecamoriformis (UTEX 1435) engineered to express a Cuphea wrightiithioesterase (CwTE2), according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the above constructsincluded different polynucleotides encoding a KASI or KASIV enzyme, 5′(SEQ ID NO: 13) and 3′ (SEQ ID NO: 14) homologous recombinationtargeting sequences (flanking the construct) to the pLoop genomic regionfor integration into the nuclear genome, and a neomycin resistanceprotein-coding sequence under the control of the C. reinhardtii(3-tubulin promoter/5′UTR (SEQ ID NO: 5) and the Chlorella vulgarisnitrate reductase 3′ UTR (SEQ ID NO: 6). This NeoR expression cassetteis listed as SEQ ID NO: 15 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to eachconstruct are listed in Table 20. The polynucleotide sequence encodingeach KAS enzyme was under the control of the P. moriformis UTEX 1435Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′UTR (SEQ ID NO: 6). The protein coding regions of the KAS enzymes andneomycin resistance gene were codon optimized to reflect the codon biasinherent in P. moriformis UTEX 1435 nuclear genes as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon transformation of individual plasmids into Strain B, positiveclones were selected on agar plates comprising G418. Individualtransformants were clonally purified and grown on sucrose as a solecarbon source at pH 7.0 under conditions suitable for lipid productionas detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of Strain B and four positive transformants of eachof pSZ2046 (Strains M-P, 1-4) are presented in Table 24.

TABLE 24 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain B. Plasmid KASI/KASIV Transit construct sourcepeptide SEQ ID NO: pSZD1134 Cuphea wrightii Native SEQ ID NO: 46 GenBankAccession No. U67317 pSZD1201 Cuphea wrightii PmSAD SEQ ID NO: 47pSZD1132 Cuphea pulcherrima Native SEQ ID NO: 48 GenBank Accession No.AAC68860 pSZD1133 Cuphea hookeriana Native SEQ ID NO: 49

TABLE 25 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainB engineered for increased C10, lauric acid, and total saturated fattyacids. Fatty Acid (Area %) Plasmid C10- % Saturates/ construct(s) No.C10 C12 C14 C16 C18:0 C18:1 C18:2 C12 Total pSZ1283 7.89 35.49 16.5811.5 1.09 19.64 6.49 43.38 72.55 pSZ1283, 1 14.94 43.97 12.19 7.56 0.7214.11 5.31 58.91 79.38 pSZD1134 pSZ1283, 2 10.27 39.61 15.35 9.61 0.9417.1 5.88 49.88 75.78 pSZD1134 pSZ1283, 3 11.69 41.83 15.21 8.77 0.8315.04 5.40 53.52 78.33 pSZD1134 D1134-20 4 10.76 40.77 15.32 9.19 0.8816.06 5.76 51.53 76.92 pSZ1283, 1 10.77 40.31 15.21 9.43 0.88 16.18 5.9751.08 76.6 pSZD1132 pSZ1283, 2 9.19 37.03 15.02 10.52 1.00 19.63 6.2946.22 72.76 pSZD1132 p5Z1283, 3 8.97 36.09 15.01 10.77 1.05 20.38 6.3945.06 71.89 pSZD1132 pSZ1283, 4 9.51 38.12 14.96 9.96 0.94 18.93 6.3247.63 73.49 pSZD1132 pSZ1283, 1 13.06 46.21 9.84 7.12 0.75 16.7 5.2259.27 76.98 pSZD1201 pSZ1283, 2 11.02 43.91 13.01 7.78 0.86 16.53 5.7754.93 76.58 pSZD1201 pSZ1283, 3 11.59 45.14 12.41 7.61 0.82 15.72 5.6556.73 77.57 pSZD1201 pSZ1283, 4 10.66 41.32 13.74 8.75 0.68 18.64 5.2151.98 75.15 pSZD1201 pSZ1283, 1 6.90 36.08 15.15 11.02 1.00 21.74 6.7742.98 70.15 pSZD1133 pSZ1283, 2 7.01 35.88 15.01 10.75 1.07 22.02 6.9342.89 69.72 pSZD1133 pSZ1283, 3 10.65 41.94 12.38 8.48 0.85 18.28 6.1552.59 74.3 pSZD1133 pSZ1283, 4 10.23 41.88 12.58 8.52 0.82 18.48 6.2252.11 74.03 pSZD1133

The data presented in Table 25 show a clear impact of the exogenousexpression of KASI and KASIV enzymes on the C10:0 and C12 fatty acidprofiles of the transformed organism. The fatty acid profiles of StrainB, expressing the Cuphea wrightii thioesterase alone, comprised about 8%C10:0 and about 35.5% C12:0, with saturated fatty acids accounting for72.55% of total fatty acids. In contrast, transformants of Strain Bengineered to additionally express a Cuphea wrightii KASI with a P.moriformis stearoyl ACP desaturase transit peptide were characterized bya fatty acid profile of about 13% C10:0 and about 46% C12:0. Saturatedfatty acids accounted for as high as 77% in transformants of Strain Bco-expressing the C. wrightii KASI fusion protein. Similarly,transformants of Strain B engineered to express the C. wrightii KASIwith the enzyme's native transit peptide were characterized by a fattyacid profile of about 15% C10, about 44% C12, and about 79% saturatedfatty acids. The fatty acid profiles or many transformants of Strain Bexpressing either Cuphea pulcherrima KASIV or Cuphea hookeriana KASIValso displayed elevated C10% and C12% levels, compared to the fatty acidprofile of Strain B itself.

These data demonstrate the utility and effectiveness of polynucleotidesenabling expression of KASI and KASIV constructs in Protothecamoriformis (UTEX 1435) to alter the percentage of saturated fatty acidsin the engineered host microbes, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids in microbial cells.

Example 38 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI Knockout

This example describes the use of recombinant polynucleotides thatdisrupt different KASI alleles to engineer microorganisms in which thefatty acid profiles of the transformed microorganisms have been enrichedin C10:0 and midchain fatty acids.

Constructs pSZ2302 and pSZ2304 were used to independently transformStrain B of Example 3, Prototheca moriformis (UTEX 1435) engineered toexpress a Cuphea wrightii thioesterase (CwTE2), according to biolistictransformation methods as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. pSZ2302 included 5′ (SEQ ID NO: 50) and 3′ (SEQ IDNO: 51) homologous recombination targeting sequences (flanking theconstruct) to the KAS 1 allele 1 genomic region for integration into theP. moriformis nuclear genome, an A. thaliana THIC protein coding regionunder the control of the C. protothecoides actin promoter/5′UTR (SEQ IDNO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). pSZ2304included 5′ (SEQ ID NO: 52) and 3′ (SEQ ID NO: 53) homologousrecombination targeting sequences (flanking the construct) to the KAS 1allele 2 genomic region for integration into the P. moriformis nucleargenome, an A. thaliana THIC protein coding region under the control ofthe C. protothecoides actin promoter/5′UTR (SEQ ID NO: 22) and C.vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This AtTHIC expressioncassette is listed as SEQ ID NO: 23 and served as a selection marker.The protein coding region of AtTHIC was codon optimized to reflect thecodon bias inherent in P. moriformis UTEX 1435 nuclear genes asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon independent transformation pSZ2302 and pSZ2304 into Strain B,thereby generating Strain Q and R, positive clones were selected on agarplates comprising thiamine Individual transformants were clonallypurified and cultivated on sucrose as a sole carbon source at pH 5.0 orpH 7.0 under heterotrophic conditions suitable for lipid production asdetailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of Strain B and positive pSZ2302 (Strain Q, 1-5) andpSZ2304 (Strain R, 1-5) transformants are presented in Tables 26 and 27.

TABLE 26 Fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains B, Q, and R engineered for increased midchain fatty acids,cultured at pH 5.0. Fatty Acid (Area %) Transformation C10- Strainplasmid(s) C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C14 U1EX 1435 None0.00 0.04 1.28 26.67 3.05 59.96 7.19 1.32 Strain B pSZ1283 0.01 0.091.09 21.60 2.21 65.15 7.94 1.19 Strain Q-1 pSZ1283, 0.08 1.21 7.52 38.711.38 38.32 8.75 8.81 pSZ2302 Strain Q-2 pSZ1283, 0.15 1.36 7.51 38.231.33 38.27 8.94 9.02 pSZ2302 Strain Q-3 pSZ1283, 0.16 1.43 7.49 38.881.30 37.58 8.73 9.08 pSZ2302 Strain Q-4 pSZ1283, 0.00 1.71 7.42 37.671.43 37.26 10.38 9.13 pSZ2302 Strain Q-5 pSZ1283, 0.13 1.21 7.36 38.811.31 38.07 8.71 8.7 pSZ2302 Strain R-1 pSZ1283, 0.19 1.78 8.47 40.111.34 33.46 9.98 10.44 pSZ2304 Strain R-2 pSZ1283, 0.90 8.00 7.78 28.961.15 30.26 17.14 16.68 pSZ2304 Strain R-3 pSZ1283, 0.26 3.58 7.77 34.981.56 32.86 14.60 11.61 pSZ2304 Strain R-4 pSZ1283, 1.64 13.50 7.61 21.380.90 36.13 14.73 22.75 pSZ2304 Strain R-5 pSZ1283, 1.03 9.63 7.56 25.611.00 31.70 18.23 18.22 pSZ2304

TABLE 27 Fatty acid profiles of Prototheca moriformis (UTEX 1435),Strains B, Q, and R engineered for increased midchain fatty acids,cultured at pH 7.0. Fatty Acid (Area %) Transformation C10- Strainplasmid(s) C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C14 ULEX None 0.010.04 1.34 27.94 3.24 57.46 7.88 1.39 1435 Strain B pSZ1283 4.72 29.5715.56 12.63 1.20 27.65 7.39 49.85 Strain Q-1 pSZ1283, pSZ2302 16.0050.61 9.52 5.33 0.54 11.79 5.28 76.13 Strain Q-2 pSZ1283, pSZ2302 16.3249.79 9.82 5.52 0.54 12.28 4.87 75.93 Strain Q-3 pSZ1283, pSZ2302 15.0847.58 10.23 5.93 0.56 15.12 4.50 72.89 Strain Q-4 pSZ1283, pSZ2302 14.2747.30 10.44 6.17 0.56 15.50 4.59 72.01 Strain Q-5 pSZ1283, pSZ2302 14.7547.28 10.32 6.04 0.59 15.50 4.65 72.35 Strain R-1 pSZ1283, pSZ2304 21.2555.42 7.97 3.65 0.00 5.46 5.66 84.64 Strain R-2 pSZ1283, pSZ2304 13.0055.05 10.88 5.78 0.28 7.90 6.29 78.93 Strain R-3 pSZ1283, pSZ2304 12.8953.15 11.11 6.13 0.00 9.87 6.13 77.15 Strain R-4 pSZ1283, pSZ2304 12.8051.64 13.86 6.69 0.00 7.51 6.70 78.3 Strain R-5 pSZ1283, pSZ2304 16.6151.42 9.84 5.27 0.33 11.15 4.79 77.87

The data presented in Tables 26 and 27 show a clear impact of disruptionof different KASI alleles on the fatty acid profiles of the transformedorganisms. When cultivated at pH 5.0, the fatty acid profiles ofPrototheca moriformis (UTEX 1435) and Prototheca moriformis (UTEX 1435)Strain B, expressing a Cuphea wrightii FATB2 thioesterase under controlof a pH regulatable promoter were very similar. These profiles werecharacterized by about 1% C14:0, about 21-26% C16:0, about 2-3% C18:0,about 60-65% C18:1, about 7% C18:2, with C10-C14 fatty acids comprisingabout 1.19-1.3% of total fatty acids. In contrast, when cultivated at pH5.0, Strain B further engineered to disrupt KASI allele 1 (Strain Q) orKASI allele 2 (Strain R) demonstrated altered fatty acid profiles thatwere characterized by increased levels of C12, increased levels of C14,decreased levels of C18, and decreased levels of C18:1 fatty acidscompared to Strain B or UTEX 1435. The fatty acid profiles of isolatesof Strains Q and R differed in that Strain R (allele 2 knockout)isolates had generally greater C12s and lower C16s and C18:1s thanStrain Q (allele 1 knockout).

When cultivated at pH 7.0, the fatty acid profile of Protothecamoriformis (UTEX 1435) is distinct from that Prototheca moriformis (UTEX1435) Strain B expressing a Cuphea wrightii FATB2 thioesterase undercontrol of a pH regulatable promoter. When cultured at pH 7.0, Strain Bwas characterized by a fatty acid profile elevated in C10, C12, and C14fatty acids (these comprised about 50% of the total fatty acids). Whencultured at pH 7.0, Strain Q and Strain R demonstrated fatty acidprofiles with still further increases in C10, C12, and C14 fatty acidsand still further decreases in C18:0 and C18:1 fatty acids relative tothat of Strain B. Again, differences in fatty acid profiles betweenStrain Q and R were observed with the profile of Strain R comprisinggreater percentage levels of C12 and lower levels of C18.1 than that ofStrain Q.

These data illustrate the successful expression and use ofpolynucleotides enabling expression of KASI and KASIV constructs inPrototheca moriformis to alter the percentage of saturated fatty acidsin the engineered host microbes, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids and decreasing theconcentration of C18:0 and C18:1 fatty acids in microbial cells. Inaddition, the data here indicate the different KASI alleles can bedisrupted to result in altered fatty acid profiles of the transformedorganisms.

Example 39 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI Knockdown

This example describes the use of recombinant polynucleotides thatencode RNA hairpins to attenuate a KASI enzyme to engineer amicroorganism in which the fatty acid profile of the transformedmicroorganism has been enriched in midchain fatty acids.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain S, was transformed independently with each of the constructspSZ2482-pSZ2485 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructspSZ2482-pSZ2485 included different polynucleotides encoding hairpin RNAstargeted against Prototheca moriformis (UTEX 1435) KASI mRNA transcriptsto down-regulate the expression of fatty acyl-ACP thioesterase, 5′ (SEQID NO: 1) and 3′ (SEQ ID NO: 2) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the nuclear genome, and a S. cerevisiae suc2 sucroseinvertase coding region (SEQ ID NO: 4) to express the protein sequencegiven in SEQ ID NO: 3 under the control of C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette islisted as SEQ ID NO: 7 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to each KASIhairpin are listed in Table 28. The polynucleotide sequence encodingeach RNA hairpin was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6). The protein coding region of the suc2 expressioncassette was codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

TABLE 28 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain S. Transformation construct Hairpin SEQ ID NO:pSZ2482 KASI hairpin B SEQ ID NO: 54 pSZ2483 KASI hairpin C SEQ ID NO:55 pSZ2484 KASI hairpin D SEQ ID NO: 56 pSZ2485 KASI hairpin E SEQ IDNO: 57

Upon independent transformation of Strain S with each of the constructslisted in Table 28, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 propagated on glucose as asole carbon source and four representative isolates of eachtransformation of Strain S, propagated on sucrose as a sole carbonsource, are presented in Table 29.

TABLE 29 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cellsengineered to express hairpin RNA constructs targeting KASI gene/geneproducts. Fatty Acid (Area %) Strain Plasmid Number C10:0 C12:0 C14:0C16:0 C18:0 C18:1 C18:2 C18:3 UTEX 1435 none 1 0.00 0.04 1.45 27.97 3.1858.35 6.78 0.60 Strain S pSZ2482 1 0.19 0.74 8.47 38.30 2.15 36.24 9.451.42 2 0.07 0.25 4.16 32.46 2.62 49.57 7.73 0.82 3 0.03 0.10 2.68 27.482.65 56.40 8.14 0.55 4 0.03 0.10 2.60 27.44 2.01 55.54 9.15 0.78 pSZ24831 0.00 0.06 1.94 30.58 1.55 53.26 9.31 0.76 2 0.20 0.05 1.76 28.01 2.3156.61 8.70 0.60 3 0.00 0.06 1.60 24.38 2.65 58.25 9.93 1.15 4 0.00 0.041.56 26.65 2.96 60.06 6.92 0.52 pSZ2484 1 0.72 3.71 19.15 38.03 1.6814.22 15.00 4.21 2 0.66 2.76 16.34 38.19 1.78 18.52 14.91 3.38 3 0.692.96 16.20 37.28 1.77 19.05 15.26 3.48 4 0.18 0.70 8.61 36.80 2.35 36.2210.89 1.10 pSZ2485 1 0.00 0.04 1.41 25.34 3.16 60.12 7.78 0.48 2 0.030.04 1.41 23.85 2.19 61.23 8.75 0.67 3 0.00 0.04 1.41 24.41 2.23 60.648.69 0.67 4 0.00 0.04 1.41 24.51 2.16 60.85 8.91 0.66

The data presented in Table 29 show a clear impact of the expression ofKAS hairpin RNA constructs on the fatty acid profiles of the transformedorganisms. The fatty acid profiles of Strain S transformants comprisingeither pSZ2482 or pSZ2484 KASI hairpin RNA construct demonstrated anincrease in the percentage of C10, C12, C14, and C16 fatty acids with aconcomitant diminution of C18:0 and C18:1 fatty acids relative to thefatty acid profile of UTEX 1435.

These data illustrate the utility and successful use of polynucleotideKASI RNA hairpin constructs in Prototheca moriformis (UTEX 1435) toalter the fatty acids profile of engineered microbes, and in particularin increasing the concentration of midchain fatty acids and decreasingC18:0 and C18:1 fatty acids in microbial cells.

Example 40 Engineering Microorganisms for Increased Production ofStearic Acid Through Fatty Acid Elongase Overexpression

This example describes the use of recombinant polynucleotides thatencode fatty acid elongases to engineer a microorganism in which thefatty acid profile of the transformed microorganism has been enriched instearic acid, arachidic acid, and docosadienoic acid.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed independently with each of the constructspSZ2323, pSZ2324, or pSZ2328 according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of theconstructs included a protein coding region to overexpress an elongase,5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2) homologous recombinationtargeting sequences (flanking the construct) to the 6S genomic regionfor integration into the nuclear genome, and a S. cerevisiae suc2sucrose invertase coding region (SEQ ID NO: 4) to express the proteinsequence given in SEQ ID NO: 3 under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitratereductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expressioncassette is listed as SEQ ID NO: 7 and served as a selectable marker.Sequence listing identities for the polynucleotides corresponding toeach elongase are listed in Table 30. The polynucleotide sequenceencoding each elongase was under control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6). The protein coding regions of the exogenous elongasesand the suc2 expression cassette were codon optimized to reflect thecodon bias inherent in P. moriformis UTEX 1435 nuclear genes asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

TABLE 30 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain J. Plasmid GenBank construct Elongase sourceAccession No. SEQ ID NO: pSZ2328 Marchantia polymorpha AAP74370 58, 59pSZ2324 Trypanosoma brucei AAX70673 60, 61 pSZ2323 Saccharomycescerevisiae P39540 62, 63

Upon independent transformation of Strain J with the constructs listedin Table 30, positive clones were selected on agar plates containingsucrose as a sole carbon source. Individual transformants were clonallypurified and propagated under heterotrophic conditions suitable forlipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 Strain J propagated onglucose as a sole carbon source and three representative isolates ofeach transformation of Strain J, propagated on sucrose as a sole carbonsource are presented in Table 31.

TABLE 31 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainJ cells engineered to overexpress elongases. Fatty Acid Area % Plasmidconstruct No C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3α C20:0 C22:2n6None 1 1.39 27.42 0.77 3.33 57.46 8.05 0.61 0.30 0.03 pSZ2328 1 1.2519.23 0.85 8.26 57.54 9.34 0.79 0.73 0.94 pSZ2328 2 1.22 17.76 0.69 7.8660.56 9.38 0.59 0.6 0.47 pSZ2328 3 1.26 18.37 0.92 7.83 58.77 10.01 0.720.64 0.52 pSZ2324 1 1.51 22.97 1.09 8.71 53.01 9.63 0.65 0.68 0.55pSZ2324 2 1.29 20.6 0.92 7.53 56.97 9.92 0.73 0.64 0.43 pSZ2324 3 1.2820.59 0.93 7.33 57.52 9.68 0.65 0.58 0.42 pSZ2323 1 1.65 27.27 0.67 3.5656.68 8.72 0.33 0.36 0.00 pSZ2323 2 1.56 28.44 0.74 3.36 55.22 9.07 0.460.39 0.03 pSZ2323 3 1.64 28.7 0.75 3.34 55.29 8.59 0.49 0.36 0.02

The data presented in Table 31 show a clear impact of the expression ofMarchantia polymorpha and Trypanosoma brucei enzymes on the C14, C16,C18:0, C20:0, and C22:2n6 fatty acid profiles of the transformedorganisms. The fatty acid profile of untransformed Strain J was about27.42% C16:0, about 3% C18:0, about 57.5% C18:1, about 0.3% C20:0 andabout 0.03% C22:2n6 fatty acids. In contrast to that of Strain J, thefatty acid profiles of Strain J transformed with different plasmidconstructs to express elongases comprised lower percentage levels of C16and higher percentage levels of C18:0, C20:0, and C22:2n6 fatty acids.The result of overexpression of Marchantia polymorpha elongase was abouta 2.5 fold increase in percentage levels of C18:0 fatty acids, a 2 foldincrease in percentage levels of C20:0 fatty acids, and about a 15 to 30fold increase in percentage levels of C22:2n6 fatty acids relative tothe fatty acid profile of Strain J.

These data illustrate the successful use of polynucleotides encodingelongases for expression in Prototheca moriformis (UTEX 1435) to alterthe fatty acid profile of engineered microbes, and in particular inincreasing the concentration of C18:0, C20:0, and C22:2n6 fatty acidsand decreasing C16:0 fatty acids in recombinant microbial cells.

Example 41 Engineering Microorganisms for Increased Production ofStearic Acid Through Acyl-ACP Thioesterase Overexpression

This example describes the use of recombinant polynucleotides thatencode different C18:0-preferring acyl-ACP thioesterases to engineermicroorganisms in which the fatty acid profiles of the transformedmicroorganisms have been enriched in stearic acid.

Classically mutagenized strains of Prototheca moriformis (UTEX 1435),Strain J or Strain A, were transformed independently with the constructslisted in Table 32 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructsincluded a protein coding region to overexpress a fatty acyl-ACPthioesterase with a C-terminal 3×FLAG® epitope tag, 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe nuclear genome, and a S. cerevisiae suc2 sucrose invertase codingregion (SEQ ID NO: 4) to express the protein sequence given in SEQ IDNO: 3 under the control of C. reinhardtii β-tubulin promoter/5′UTR (SEQID NO: 5) and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO:6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO:7 and served as a selectable marker. Sequence listing identities for thepolynucleotides corresponding to each thioesterase are listed in Table32. The polynucleotide sequence encoding each thioesterase was undercontrol of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C.vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). The protein codingregions of the exogenous thioesterases and the suc2 expression cassettewere codon optimized to reflect the codon bias inherent in P. moriformisUTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

TABLE 32 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain A or Strain J. Acyl-ACP Thioesterase, Acyl-ACPTransit Plasmid GenBank Thioesterase Peptide construct Accession No.source source SEQ ID NO: pSZD581 FATA, Brassica napus native 64, 65CAA52070 pSZD643 FATA, Brassica napus UTEX 66, 67 CAA52070 250 SADpSZD645 FATA, C. tinctorius UTEX 68, 69 AAA33019 250 SAD pSZD644 FATA,Ricinis communis native 70, 71 ABS30422 pSZD1323 FATA, G. mangostananative 72, 73 AAB51523 pSZD1320 FATA Theobroma cacao native 74, 75

Upon independent transformation of Strain A or J with the constructslisted in Table 32, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 Strain J propagated onglucose as a sole carbon source and representative isolates of eachtransformation of Strain J, propagated on sucrose as a sole carbonsource are presented in Table 33.

TABLE 33 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainJ cells engineered to overexpress exogenous acyl-ACP thioesteraseenzymes. Plasmid Fatty Acid Area % Strain construct No. C14:0 C16:0C18:0 C18:1 C18:2 C18:3α A None 1 1.08 25.48 3.23 59.70 8.25 0.70 J None1 1.41 27.33 3.38 57.07 8.15 0.64 A pSZD581 1 1.02 26.60 14.47 44.8010.05 0.65 2 1.08 28.24 13.57 43.89 10.07 0.68 3 0.97 24.70 9.13 50.8511.27 0.82 A pSZD643 1 1.39 26.97 16.21 44.10 8.43 0.83 2 1.37 27.9111.15 48.31 8.40 0.78 A pSZD645 1 0.90 23.39 8.35 50.69 13.34 0.96 ApSZD644 1 1.67 19.70 4.40 59.15 12.32 1.01 J pSZD1323 1 1.33 23.26 9.2853.42 10.35 0.69 2 1.47 26.84 7.36 52.78 9.29 0.64 3 1.43 26.31 6.0554.45 9.37 0.66 J pSZD1320 1 1.30 24.76 3.84 60.90 6.96 0.55 2 1.3626.30 3.27 58.19 8.66 0.48 3 1.39 25.51 3.18 58.78 8.85 0.45

The data presented in Table 33 show a clear impact of the expression ofexogenous acyl-ACP enzymes on the fatty acid profiles of the transformedmicroorganisms. The fatty acid profiles of untransformed Strain A and Jwere about 25% C16:0, about 3.3% C18:0, about 57 to 60% C18:1. Incontrast, the fatty acid profiles of Strain A transformed with differentplasmid constructs to express acyl-ACP enzymes comprised greaterpercentage levels of C18:0 and lower percentage levels of C18:1 fattyacids than that of Strain A. Expression of FATA enzymes from B. napus,C. tinctorius, R. communis and G. mangostana in Strain A or J enabledthe accumulation of stearate levels in the transformed organisms. Theresult of overexpression of a Brassica napus acyl-ACP thioesterase wasabout a 2 to 5 fold increase in the percentage levels of C18:0 fattyacids of the fatty acid profile of the transformed organisms relative tothe fatty acid profile of Strain A. Fatty acid profiles of cellsengineered to overexpress a G. mangostana acyl-ACP FATA thioesterasewith a C. protothecoides SAD1 transit peptide were characterized byabout a 2 to 3 fold increase in the percentage levels of C18:0 fattyacids of the fatty acid profile of the transformed organisms relative tothe fatty acid profile of Strain J.

These data illustrate the utility and effective use of polynucleotidesencoding fatty acyl-ACP thioesterases for expression in Protothecamoriformis (UTEX 1435) to alter the fatty acid profile of engineeredmicrobes, and in particular in increasing the concentration of C18:0 anddecreasing C18:1 fatty acids in recombinant microbial cells.

Example 42 Engineering Microorganisms for Increased Production of ErucicAcid Through Elongase or Beta-Ketoacyl-CoA Synthase Overexpression

In an embodiment of the present invention, a recombinant polynucleotidetransformation vector operable to express an exogenous elongase orbeta-ketoacyl-CoA synthase in an optionally plastidic oleaginous microbeis constructed and employed to transform Prototheca moriformis (UTEX1435) according to the biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696 to obtain a cell increased forproduction of erucic acid. The transformation vector includes a proteincoding region to overexpress an elongase or beta-ketoacyl-CoA synthasesuch as those listed in Table 5, promoter and 3′UTR control sequences toregulate expression of the exogenous gene, 5′ and 3′ homologousrecombination targeting sequences targeting the recombinantpolynucleotides for integration into the P. moriformis (UTEX 1435)nuclear genome, and nucleotides operable to express a selectable marker.The protein-coding sequences of the transformation vector arecodon-optimized for expression in P. moriformis (UTEX 1435) as describedin PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Recombinant polynucleotidesencoding promoters, 3′ UTRs, and selectable markers operable forexpression in P. moriformis (UTEX 1435) are disclosed herein and inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon transformation of the transformation vector into P. moriformis(UTEX 1435) or a classically-mutagenized strain of P. moriformis (UTEX1435), positive clones are selected on agar plates. Individualtransformants are clonally purified and cultivated under heterotrophicconditions suitable for lipid production as detailed inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples are preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples are analyzed using fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. As a result of these manipulations, the cell mayexhibit an increase in erucic acid of at least 5, 10, 15, or 20 fold.

Example 43 Generation of Capric, Lauric, and Myristic Acid Rich Oils inStrain Utex1435 by the Expression of Cuphea PSR23 LPAATs

We tested the effect of expression of two 1-acyl-sn-glycerol-3-phosphateacyltransferases (LPAATs) in a previously described P. moriformis (UTEX1435) transgenic strain, expressing the acyl ACP thioesterase (FATB2)from Cuphea wrightii. The LPAAT2 and LPAAT3 genes from Cuphea PSR23(CuPSR23) were identified by analysis of a combination of CuPSR23genomic sequences and transcriptomic sequences derived from seed RNAs.The two LPAATs have not been previously described. The genes were codonoptimized to reflect UTEX 1435 codon usage. Transformations, cellculture, lipid production and fatty acid analysis were all carried outas previously described.

Increased capric, lauric, and myristic accumulation in strain B by theexpression of the Cuphea PSR23 1-acyl-sn-glycerol-3-phosphateacyltransferases (LPAAT2 and LPAAT3) [pSZ2299 and pSZ2300,respectively]: In this example, transgenic strains were generated viatransformation of strain B with the constructs pSZ2299 or pSZ2300,encoding CuPSR23 LPAAT2 and LPAAT3, respectively. The transgenic strainswere selected for resistance to the antibiotic G418. Construct pSZ2299can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT2-1:CvNR::pLOOP3′.Construct pSZ2300 can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT3-1:CvNR::pLOOP3′. Thesequence of the transforming DNA (pSZ2299 and pSZ2300) is providedbelow. The relevant restriction sites in the construct from 5′-3′,BspQI, KpnI, XbaI, Mfe I, BamHI, EcoRI, SpeI, XhoI, SacI, BspQI,respectively, are indicated in lowercase, bold, and underlined. BspQIsites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences at the 5′ and 3′ end of the construct representgenomic DNA from UTEX 1435 that target integration to the pLoop locusvia homologous recombination. Proceeding in the 5′ to 3′ direction, theselection cassette has the C. reinhardtii β-tubulin promoter drivingexpression of the NeoR gene (conferring resistance to G418) and theChlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. The promoter isindicated by lowercase, boxed text. The initiator ATG and terminator TGAfor NeoR are indicated by uppercase italics, while the coding region isindicated with lowercase italics. The 3′ UTR is indicated by lowercaseunderlined text. The spacer region between the two cassettes isindicated by upper case text. The second cassette containing the codonoptimized LPAAT2 gene (pSZ2299) or LPAAT3 gene (pSZ2300) from CupheaPSR23 is driven by the Prototheca moriformis endogenous AMT3 promoter,and has the same Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR.In this cassette, the AMT3 promoter in indicated by lowercase, boxedtext. The initiator ATG and terminator TGA for the CuPSR23 LPAAT2 andLPAAT3 genes are indicated in uppercase italics, while the codingregions are indicated by lowercase italics. The 3′ UTR is indicated bylowercase underlined text. The final constructs were sequenced to ensurecorrect reading frames and targeting sequences.

pSZ2299 Transforming Construct: (SEQ ID NO: 90) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggt

cctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatccCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGG

ctagtATGgcgatcgcggccgcggcggtgatcttcctgttcggcctgatcttcttcgcctccggcctgatcatcaacctgttccaggcgctgtgcttcgtcctgatccgccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgcggagctgctgctgtccgagctgctgtgcctgttcgactggtgggcgggcgcgaagctgaagctgttcaccgaccccgagacgttccgcctgatgggcaaggagcacgccctggtcatcatcaaccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctccatcatctccgtcgccaagaagtccacgaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacctggagcgctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgatcgactaccccctgcccttctggctggtcatcttcgtcgagggcacccgcttcacgcgcacgaagatgctggcggcccagcagtacgcggtctcctccggcctgcccgtcccccgcaacgtcctgatcccccgcacgaagggcttcgtctcctgcgtgtcccacatgcgctccttcgtccccgcggtgtacgacgtcacggtggcgttccccaagacgtcccccccccccacgctgctgaacctgttcgagggccagtccatcatgctgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtcgcggagtggtgccgcgacaagttcgtcgagaaggacgccctgctggacaagcacaacgcggaggacacgttctccggccaggaggtgtgccactccggctcccgccagctgaagtccctgctggtcgtgatctcctgggtcgtggtgacgacgttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggcgttctccgccatcggcctgggcatcgtcaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccgaggtggcccaggccaagctgaagaccggcctgtccatctccaagaaggtgacggacaaggagaacTGAttaattaactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctta gctcttcgpSZ2300 Transforming Construct: (SEQ ID NO: 91) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggt

cctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgattcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatccCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGG

ctagtATGgccatcgcggcggccgcggtgatcgtgcccctgtccctgctgttcttcgtgtccggcctgatcgtcaacctggtgcaggccgtctgcttcgtcctgatccgccccctgtccaagaacacgtaccgccgcatcaaccgcgtggtcgcggagctgctgtggctggagctggtgtggctgatcgactggtgggcgggcgtgaagatcaaggtcttcacggaccacgagacgttccacctgatgggcaaggagcacgccctggtcatctgcaaccacaagtccgacatcgactggctggtcggctgggtcctgggccagcgctccggctgcctgggctccaccctggcggtcatgaagaagtcctccaagttcctgcccgtcctgggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagatcacgctgaagtccggcctgaaccgcctgaaggactaccccctgcccttctggctggcgctgttcgtggagggcacgcgcttcacccgcgcgaagctgctggcggcgcagcagtacgccgcgtcctccggcctgcccgtgccccgcaacgtgctgatcccccgcacgaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgcgatctacgacgtcaccgtggccatccccaagacgtcccccccccccacgctgatccgcatgttcaagggccagtcctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgccgtcgcgcagtggtgccgcgacatcttcgtggagaaggacgcgctgctggacaagcacaacgccgaggacaccttctccggccaggagctgcaggagaccggccgccccatcaagtccctgctggtcgtcatctcctgggccgtcctggaggtgttcggcgccgtcaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggcgttctccggcatcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggccc

cagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctcttcg

To determine the impact of the CuPSR23 LPAAT2 and LPAAT3 genes onmid-chain fatty acid accumulation, the above constructs containing thecodon optimized CuPSR23 LPAAT2 or LPAAT3 genes driven by the UTEX 1453AMT3 promoter were transformed into strain B.

Primary transformants were clonally purified and grown under standardlipid production conditions at pH 7.0 (all the strains require growth atpH 7.0 to allow for maximal expression of the CuPSR23 LPAAT2 or LPAAT3gene driven by the pH-regulated AMT3 promoter). The resulting profilesfrom a set of representative clones arising from these transformationsare shown in Table 34, below. D1520 represents clones of Strain B withCuPSR23 LPAAT2 and D1521 represents clones of Strain B with CuPSR23LPAAT3.

TABLE 34 Fatty acid profiles of Strain B and representative transgeniclines transformed with pSZ2299 and pSZ2300 DNA. Sample ID C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 Strain B 4.83 28.54 15.64 12.64 1.3 27.997.75 Sample ID C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 D1520-A 8.5935.09 16.55 11.96 1.69 19.49 5.59 D1520-B 8.13 33.93 16.46 12.44 1.5720.66 5.96 D1520-C 7.6 33.1 16.21 12.65 1.5 21.41 6.48 D1520-D 7.3532.54 16.03 12.79 1.67 22.16 6.41 D1520-E 7.28 32.21 16.2 12.99 1.7322.39 6.28 Sample ID C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 D1521-A6.14 31.5 15.98 12.96 1.96 22.52 8 D1521-B 6.17 31.38 15.98 12.87 2.0822.54 7.92 D1521-C 5.99 31.31 15.75 12.79 2.23 22.45 8.36 D1521-D 5.9531.05 15.71 12.84 2.48 22.69 8.32 D1521-E 5.91 30.58 15.85 13.22 1.9723.55 7.84

The transgenic CuPSR23 LPAAT2 strains (D1520A-E) show a significantincrease in the accumulation of C10:0, C12:0, and C14:0 fatty acids witha concomitant decrease in C18:1 and C18:2. The transgenic CuPSR23 LPAAT3strains (D1521A-E) show a significant increase in the accumulation ofC10:0, C12:0, and C14:0 fatty acids with a concomitant decrease inC18:1. The expression of the CuPSR23 LPAAT in these transgenic linesappears to be directly responsible for the increased accumulation ofmid-chain fatty acids in general, and especially laurates. While thetransgenic lines show a shift from longer chain fatty acids (C16:0 andabove) to mid-chain fatty acids, the shift is targeted predominantly toC10:0 and C12:0 fatty acids with a slight effect on C14:0 fatty acids.The data presented also show that co-expression of the LPAAT2 and LPAAT3genes from Cuphea PSR23 and the FATB2 from C. wrightii (expressed in thestrain Strain B) have an additive effect on the accumulation of C12:0fatty acids.

Our results suggest that the LPAAT enzymes from Cuphea PSR23 are activein the algal strains derived from UTEX 1435. These results alsodemonstrate that the enzyme functions in conjunction with theheterologous FatB2 acyl-ACP thioesterase enzyme expressed in Strain B,which is derived from Cuphea wrightii.

Example 44 Alteration of Fatty Acid Levels in Strain Utex1435 by theExpression of Cuphea PSR23 LPAATX in Combination with Cuphea wrighthFATB2

Here we demonstrate the effect of expression of a1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) in a previouslydescribed P. moriformis (UTEX 1435) transgenic strain, Strain B. Asdescribed above, Strain B is a transgenic strain expressing the acyl ACPthioesterase (FATB2) from Cuphea wrightii, which accumulates C12:0 fattyacids between 40 to 49%. Further to Example 43, a third CuPSR23 LPAAT,LPAATx, was identified by analysis of a combination of CuPSR23 genomicsequences and transcriptomic sequences derived from seed RNAs.Expression of a mid-chain specific LPAAT should thus increase thepercentage of TAGs that have a capric acid (C10:0 fatty acid), lauricacid (C12:0 fatty acid), or myristic acid (C14:0 fatty acid) at the sn-2position, and should consequently elevate the overall levels of thesefatty acids. In Example 43, LPAAT2 and LPAAT3 were shown to increasecaprate, laurate, and myristate accumulation in strain B. LPAATx wasintroduced into strain B to determine its effect on fatty acid levels inthis strain. The LPAATx gene was codon optimized to reflect UTEX 1435codon usage. Transformations, cell culture, lipid production and fattyacid analysis were all carried out as previously described.

Decreased Caprate, Laurate, and Myristate Accumulation and IncreasedPalmitate and Stearate Accumulation in Strain Strain B by the Expressionof the Cuphea PSR23 1-acyl-sn-glycerol-3-phosphate Acyltransferase(LPAATx) [pSZ2575]:

In this example, transgenic strains were generated via transformation ofstrain B with the construct pSZ2575 encoding CuPSR23 LPAATx. Thetransgenic strains were selected for resistance to the antibiotic G418.Construct pSZ2575 can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAATx:CvNR::pLOOP3′. Thesequence of the transforming DNA is provided below (pSZ2575). Therelevant restriction sites in the construct from 5′-3′, BspQ1, KpnI,XbaI, MfeI, BamHI, EcoRI, SpeI, XhoI, SacI, BspQ1, respectively, areindicated in lowercase, bold, and underlined. BspQ1 sites delimit the 5′and 3′ ends of the transforming DNA. Bold, lowercase sequences at the 5′and 3′ end of the construct represent genomic DNA from UTEX 1435 thattarget integration to the pLoop locus via homologous recombination.Proceeding in the 5′ to 3′ direction, the selection cassette has the C.reinhardtii β-tubulin promoter driving expression of the NeoR gene(conferring resistance to G418) and the Chlorella vulgaris NitrateReductase (NR) gene 3′ UTR. The promoter is indicated by lowercase,boxed text. The initiator ATG and terminator TGA for NeoR are indicatedby uppercase italics, while the coding region is indicated withlowercase italics. The 3′ UTR is indicated by lowercase underlined text.The spacer region between the two cassettes is indicated by upper casetext. The second cassette containing the codon optimized LPAATx gene(pSZ2575) from Cuphea PSR23 is driven by the Prototheca moriformisendogenous AMT3 promoter, and has the same Chlorella vulgaris NitrateReductase (NR) gene 3′ UTR. In this cassette, the AMT3 promoter isindicated by lowercase, boxed text. The initiator ATG and terminator TGAfor the CuPSR23 LPAATx genes are indicated in uppercase italics, whilethe coding region is indicated by lowercase italics. The 3′ UTR isindicated by lowercase underlined text. The final construct wassequenced to ensure correct reading frame and targeting sequences.

pSZ2575 Transforming Construct: (SEQ ID NO: 92) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggt

cctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatccCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGG

ctagtATGgagatccccccccactgcctgtgctccccctcccccgccccctcccagctgtactacaagaagaagaagcacgccatcctgcagacccagaccccctaccgctaccgcgtgtcccccacctgcttcgcccccccccgcctgcgcaagcagcacccctaccccctgcccgtgctgtgctaccccaagctgctgcacttctcccagccccgctaccccctggtgcgctcccacctggccgaggccggcgtggcctaccgccccggctacgagctgctgggcaagatccgcggcgtgtgcttctacgccgtgaccgccgccgtggccctgctgctgttccagtgcatgctgctgctgcaccccttcgtgctgctgttcgaccccttcccccgcaaggcccaccacaccatcgccaagctgtggtccatctgctccgtgtccctgttctacaagatccacatcaagggcctggagaacctgccccccccccactcccccgccgtgtacgtgtccaaccaccagtccttcctggacatctacaccctgctgaccctgggccgcaccttcaagttcatctccaagaccgagatcttcctgtaccccatcatcggctgggccatgtacatgctgggcaccatccccctgaagcgcctggactcccgctcccagctggacaccctgaagcgctgcatggacctgatcaagaagggcgcctccgtgttcttcttccccgagggcacccgctccaaggacggcaagctgggcgccttcaagaagggcgccttctccatcgccgccaagtccaaggtgcccgtggtgcccatcaccctgatcggcaccggcaagatcatgccccccggctccgagctgaccgtgaaccccggcaccgtgcaggtgatcatccacaagcccatcgagggctccgacgccgaggccatgtgcaacgaggcccgcgccaccatctcccactccctggacgacTGAttaattaactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctta g ctcttcg

To determine the impact of the CuPSR23 LPAATx gene on fatty acidaccumulation, the above construct containing the codon optimized CuPSR23LPAATx gene driven by the UTEX 1453 AMT3 promoter was transformed intostrain B.

Primary transformants were clonally purified and grown under lownitrogen conditions at pH 7.0; the strains require growth at pH 7.0 toallow for maximal expression of the CuPSR23 LPAATx and CwFATB2 genesdriven by the pH-regulated AMT3 promoter. The resulting profiles from aset of representative clones arising from these transformations areshown in Table 35, below. D1542 represents clones of Strain B withCuPSR23 LPAATx.

TABLE 35 Fatty acid profiles of Strain B and representative transgeniclines transformed with pSZ2575. Sample ID C10:0 C12:0 C14:0 C16:0 C18:0C18:1 C18:2 Strain B 4.77 28.63 15.48 12.65 1.28 28.20 7.57 Sample IDC10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 D1542-A 1.19 13.25 10.48 21.344.49 32.07 14.78 D1542-B 1.15 14.01 10.62 20.61 3.99 32.12 15.24 D1542-C1.21 13.69 10.83 20.40 3.59 33.54 15.05 D1542-D 1.56 16.83 11.51 18.442.94 33.97 12.74 D1542-E 2.15 18.58 11.94 18.22 3.17 32.63 1.62

The transgenic CuPSR23 LPAATx strains (D1542A-E) show a significantdecrease in the accumulation of C10:0, C12:0, and C14:0 fatty acidsrelative to the parent, Strain B, with a concomitant increase in C16:0,C18:0, C18:1 and C18:2. The expression of the CuPSR23 LPAATx gene inthese transgenic lines appears to be directly responsible for thedecreased accumulation of mid-chain fatty acids (C10-C14) and theincreased accumulation of C16:0 and C18 fatty acids, with the mostpronounced increase observed in palmitates (C16:0). The data presentedalso show that despite the expression of the midchain specific FATB2from C. wrightii (present in Strain B), the expression of CuPSR23 LPAATxappears to favor incorporation of longer chain fatty acids into TAGs.

Our results suggest that the LPAATx enzyme from Cuphea PSR23 is activein the algal strains derived from UTEX 1435. Contrary to Cuphea PSR23LPAAT2 and LPAAT3, which increase mid-chain fatty acid levels, CuPSR23LPAATx leads to increased C16:0 and C18:0 levels. These resultsdemonstrate that the different LPAATs derived from CuPSR23 (LPAAT2,LPAAT3, and LPAATx) exhibit different fatty acid specificities in StrainB as judged by their effects on overall fatty acid levels.

Example 45 Reduction in Chain Length of Fatty Acid Profile as a Resultof Overexpressing an Endogenous Microalgal FATA Acyl-ACP Thioesterase

Here, we demonstrate that over expression of the Prototheca moriformisendogenous thioesterases FATA1 in UTEX1435 results in a clear diminutionof cell triglyceride C18:0 and C18:1 acyl chains with an increase inC16:0, C14:0.

Constructs used for the over expression of the P. moriformis FATA1 gene(pSZ2422, pSZ2421): To over express the PmFATA1 in P. moriformis STRAINJ, a codon optimized PmFATA1 gene was been transformed into STRAIN J.The Saccharomyces cerevisiae invertase gene was utilized as theselectable marker to confer the ability of growing on sucrose media. Theconstruct pSZ2422 that have been expressed in STRAIN J can be writtenas: 6SA:: CrTUB2-ScSUC2-CvNR3′:PmAMT3-Pm FATA1 (opt)-CvNR3′::6SB, andthe construct pSZ2421 can be written as 6SA::CrTUB2-ScSUC2-CvNR3′:PmAMT3-S106SAD TP-Pm FATA1 (opt)-CvNR3′::6SB.

The sequence of the transforming DNA is provided below. Relevantrestriction sites in the construct pSZ2422 are indicated in lowercase,bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, Mfe I, BamH I,EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sitesdelimit the 5′ and 3′ ends of the transforming DNA. Bold, lowercasesequences represent genomic DNA from STRAIN J that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof STRAIN J to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of P. moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the PmFATA1 are indicated byuppercase, bold italics, while the remainder of the gene is indicated bybold italics. The C. vulgaris nitrate reductase 3′ UTR is againindicated by lowercase underlined text followed by the STRAIN J 6Sgenomic region indicated by bold, lowercase text.

Relevant restriction sites in the construct pSZ2421 are the same aspSZ2422. In pSZ2421, the PmFATA1 is fused to the Chlorellaprotothecoides S106 stearoyl-ACP desaturase transit peptide and thetransit peptide is located between initiator ATG of PmFATA1 and the AscI site.

Nucleotide sequence of transforming DNA contained in pSZ2422:(SEQ ID NO: 93) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccdcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcc

cgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtagggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgac

ggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatg

acttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctc ttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

To determine the impact on fatty acid profiles when the endogenous FATA1gene have been over expressed in STRAIN J, both the P. moriformis FATA1with native transit peptide and PmFATA1 fused to a Chlorellaprotothecoides SAD transit peptide were driven by the amt03 promoter andthe resulting plasmids were transformed independently into STRAIN J.

Primary transformants were clonally purified and grown underlow-nitrogen lipid production conditions at pH 7.0 (all the plasmidsrequire growth at pH 7.0 to allow for maximal PmFATA1 gene expressionwhen driven by the pH regulated amt03 promoter). The resulting profilesfrom representative clones arising from transformations with pSZ2422 andpSZ2421 into STRAIN J are shown in the tables below.

In Table 36, below, the impact of over expressing native PmFATA1 is aclear diminution of C18:1 chain lengths with an increase in C16:0,C14:0, and possibly in C18:0. Considering the protein localization ofprocessing, we also tried the PmFATA1 fused to a Chlorellaprotothecoides stearoyl-ACP desaturase transit peptide. Similar to theresults we observed in the amt03-native PmFATA1 construct, the C16:0 andC14:0 levels are significantly higher than the parental strain J.

TABLE 36 Fatty acid profiles in Strain J and derivative transgenic linestransformed with pSZ2422 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH7; Strain J; T374; 7.69 55.00 4.92 24.94 5.19 D1377-7 96well pH 7;Strain J; T374; 6.39 54.11 5.85 25.91 5.76 D1377-13 96well pH 7; StrainJ; T374; 6.57 53.55 4.68 27.18 5.74 D1377-14 96well pH 7; Strain J;T374; 5.29 49.93 4.24 30.76 7.27 D1377-16 96well pH 7; Strain J; T374;4.76 49.10 4.75 32.36 6.77 D1377-9 96well pH 7; Strain J; T374; 4.2846.06 5.14 35.87 6.69 D1377-19 96well Ctrl-pH 7; Strain J 1.42 27.633.31 57.20 8.00

TABLE 37 Fatty acid profiles in STRAIN J and derivative transgenic linestransformed with pSZ2421 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH7; STRAIN J; T374; 6.76 57.06 4.12 23.66 6.07 D1376-21 96well pH 7;STRAIN J; T374; 6.56 54.62 5.44 25.69 5.64 D1376-22 96well pH 7; STRAINJ; T374; 4.54 48.38 4.27 33.23 7.24 D1376-23 96well pH 7; STRAIN J;T374; 4.48 47.66 4.60 34.28 6.91 D1376-19 96well pH 7; STRAIN J; T374;4.53 47.30 4.67 34.51 6.80 D1376-20 96well pH 7; STRAIN J; T374; 3.5642.70 4.03 39.85 7.52 D1376-17 96well Ctrl-pH 7; STRAIN J 1.42 27.633.31 57.20 8.00

Thus, we conclude that percent myristic and lauric acid levels in thefatty acid profile of a microalgal cell can be increased byoverexpression of a C18-preferring acyl-ACP thioesterase.

Example 46 Natural Oils Suitable for Use as Roll-in Shortenings

The nutritional and functional properties of edible fats have beentraditionally associated with specific chemical compositions andcrystallization conditions. Switching from one oil source to another isusually a difficult task since both the melting behavior and structureof the fat changes dramatically, leading to adverse changes infunctionality. In recent history, we can recall the painful period whenpartially hydrogenated fats were replaced with palm oil and palm oilfractions. We examined how the yield stress, elastic modulus,polymorphism, microstructure and melting profile of two fats with vastlydifferent chemical compositions can be matched. Oil A was produced fromPrototheca moriformis cells expressing an exogenous invertase and anUlmus americana acyl-ACP thioesterase with a Chlorella protothecoidesplastid targeting sequence. Oil B was produced from Protothecamoriformis cells expressing an exogenous invertase and a Cupheahookeriana acyl-ACP thioesterase. Oil A contained greater than 62% (w/w)medium chain fatty acids, or MCT (C8:0-C14:0), 23% (C16:0+C18:0) and 9%C18:1, while Oil B contained less than 2% C8:0-C14:0, 54% (C16:0+C18:0)and 29% C18:1. Oil A was thus a medium chain triglyceride rich fat,while Oil B resembled palm oil. Both oils had a solid fat content of˜45% at 20° C., and very similar SFC versus temperature profiles. DSC(dynamic scanning calorimetry) melting profiles showed two major peakscentered around ˜12-13° C. and ˜28-35° C. Both fats were in thebeta-prime polymorphic form (as determined by X-ray diffraction) anddisplayed asymmetric, elongated crystallite morphology withcharacteristic features. The yield stresses and storage moduli (G′) ofOil A and Oil B were 520-550 Pa, and 7×10⁶ Pa-1.8×10⁷ Pa, respectively.A yield stress in this region suggests a satisfactory plasticity, whichcombined with a high storage modulus makes for an ideal roll-inshortening. Thus, it is possible to alter the chemical composition of afood oil while retaining its lamination functionality.

Other suitable enzymes for use with the cells and methods of any of theabove embodiments of the invention include those that have at least 70%amino acid identity with one of the proteins listed in the descriptionabove and that exhibit the corresponding desired enzymatic activity. Inadditional embodiments, the enzymatic activity is present in a sequencethat has at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 99% identity withone of the above described nucleic acid sequences, all of which arehereby incorporated by reference as if fully set forth.

Example 47 Fractionation to Remove Trisaturates from a TailoredMicrobial Oil that is a Cocoa Butter Mimetic

A refined bleached and deodorized oil was obtained from Strain K4 (seeExample 35). The oil was heated to 70° C. and cooled at 0.5° C. per minto 36° C. and held at 36° C. for 1 hour. An approximately 2.5 ml samplewas then centrifuged at 36° C. for 1 hour at 4300. A liquid supernatantwas recovered and analyzed using lipase and mass spectrometry. Thesample was found to be depleted in tristearin (SSS), SSP, and PPS. Thetriacylglycerols of the sample were found to be very similar to that ofcocoa butter and the liquid supernatant was even closer to that of cocoabutter in terms of low amounts of trisaturates.

TABLE 38 TAG profile of oil from the K4 strain before and afterfractionation as compared to cocoa butter. fractionation upper TAG K4oil layer (liquid) cocoa butter OOL (+?) 0.12 0.12 0.00 POL 0.23 0.310.33 PLP 2.41 3.38 1.58 MOP 0.93 1.25 0.00 PPM (+MMS) 0.42 0.29 0.00 OOO0.23 0.34 0.00 SOL 0.36 0.47 0.32 OOP 0.95 1.42 2.44 PLS 5.66 7.90 2.90POP (+MSO) 11.80 15.20 17.93 PPP + MPS 2.22 1.07 0.36 OOS 1.19 1.68 3.02SLS (+PLA) 3.96 5.11 1.77 POS 27.22 32.80 40.25 PPS (+SSM) 6.47 1.520.49 MaOO 0.00 0.00 0.36 SLA 0.31 0.34 0.00 SOS (+POA) 17.84 22.50 24.93SSP (+PPA) 9.24 0.96 0.63 SOA (+POB) 1.39 1.68 1.51 SSS (+PSA) 5.25 0.230.33 SOB + LgOP 0.38 0.44 0.27 SSA 0.41 0.00 0.00 SOLg 0.41 0.00 0.00PSLg + ASB 0.26 0.00 0.00 SOHx 0.12 0.51 0.00 SSLg 0.21 0.14 0.15 SUMarea % 100.00 99.67 99.57

Example 48 Production of High-Stearate Triglyceride Oil in an OleaginousCell by Overexpression of KASII, Knockout of One SAD Allele andRepression of a Second SAD Allele

The oleaginous, non-photosynthetic alga, Prototheca moriformis, storescopious amounts of triacylglyceride oil under conditions where thenutritional carbon supply is in excess, but cell division is inhibiteddue to limitation of other essential nutrients. Bulk biosynthesis offatty acids with carbon chain lengths up to C18 occurs in the plastids;fatty acids are then exported to the endoplasmic reticulum whereelongation past C18 and incorporation into triacylglycerides (TAGs) isbelieved to occur. Lipids are stored in large cytoplasmic organellescalled lipid bodies until environmental conditions change to favorgrowth, whereupon they are rapidly mobilized to provide energy andcarbon molecules for anabolic metabolism. Wild-type P. moriformisstorage lipid is mainly comprised of ˜60% oleic (C18:1), ˜25-30%palmitic (C16:0), and ˜5-8% linoleic (C18:2) acids, with minor amountsof stearic (C18:0), myristic (C14:0), α-linolenic (C18:3α), andpalmitoleic (C16:1) acids. This fatty acid profile results from therelative activities and substrate affinities of the enzymes of theendogenous fatty acid biosynthetic pathway. P. moriformis is amenable tomanipulation of fatty acid and lipid biosynthesis using moleculargenetic tools, enabling the production of oils with fatty acid profilesthat are very different to the wild-type composition. Herein we describestrains where we have modified the expression of stearoyl-ACP desaturase(SAD) and β-ketoacyl-ACP synthase II (KASII) genes in order to generatestrains with up to 57% stearate and as little as 7% palmitate. Weidentify additional strains with up to 55% stearate and as low as 2.4%linoleate when we perform similar modifications in conjunction withdown-regulating the expression of the FATA thioesterase and the FAD2fatty acid desaturase genes.

Soluble SADs are plastid-localized, di-iron enzymes which catalyze thedesaturation of acyl carrier protein (ACP)-bound stearate to oleate(C18:1 cis-Δ⁹). Previously, we have established that hairpin constructstargeting the SAD1 or SAD2 transcripts activate the cellular RNAinterference (RNAi) machinery, down-regulating SAD activity andresulting in elevated levels of C18:0 in the storage lipid. SAD activityis also reduced in strains where we disrupt one of the two alleles ofSAD2, encoding the major SADs that are expressed during storage lipidbiosynthesis. The Fatty Acid Desaturase 2 (FAD2) gene encodes anendoplasmic reticulum membrane-associated desaturase that convertsoleate to linoleate (C18:2 cis-Δ⁹, cis-Δ¹²).) Hairpin RNAi constructstargeting FAD2 reduce linoleate levels to 1-2%. KASII is a fatty acidsynthase which specifically catalyzes the condensation of malonyl-ACPwith palmitoyl (C16:0)-ACP to form β-keto-stearoyl-ACP. We have shownthat overexpression of KASII in P. moriformis causes C16:0 levels todecrease with a concomitant increase in C18:1 abundance. In the examplesbelow we demonstrate that by down-regulating SAD gene expression usingRNAi, disrupting an allele of the SAD2 gene, and overexpressing theKASII fatty acid synthase, we generate strains capable of accumulatingstearate in excess of 50% of the total fatty acids, and with SOS as themajor TAG species. SOS levels increase up to 47% in strains whichcombine SAD2 and FAD2 down-regulation with KASII overexpression.

Constructs Used for SAD2 Knockout/RNAi in Strain J:

A DNA construct, pSZ2282, was made to simultaneously disrupt the SAD2-1allele and express a SAD2 hairpin construct in Strain J. A Saccharomycescerevisiae SUC2 gene, encoding sucrose invertase, which wascodon-optimized for expression in P. moriformis, was utilized as aselectable marker for transformation. The sequence of the transformingDNA is provided immediately below. Relevant restriction sites areindicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, AscI,MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI,respectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Underlined sequences at the 5′ and 3′ flanks of the constructrepresent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the Chlamydomonasreinhardtii TUB2 promoter driving the expression of the Saccharomycescerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, therebypermitting the strain to grow on sucrose) is indicated by lowercase,boxed text. The initiator ATG and terminator TGA for SUC2 are indicatedby uppercase italics, while the coding region is indicated withlowercase italics. The 3′ UTR of the Chlorella vulgaris nitratereductase (NR) gene is indicated by small capitals, followed by a spacerregion indicated by lowercase text. A second C. reinhardtii TUB2promoter sequence, indicated by lowercase boxed text, drives expressionof the SAD2 hairpin C sequence. The sense and antisense strands areindicated with uppercase, bold italics, and are separated by the P.moriformis Δ ¹²-fatty acid desaturase (FAD2) intron and the first 10bases of the FAD2 second exon (uppercase italics). A second C. vulgarisNR 3′ UTR is indicated by small capitals.

Nucleotide sequence of the transforming DNA from pSZ2282:(SEQ ID NO: 94) gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaacaacccttattt

gaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctagggatatc

CAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGgagctccagccacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatgatgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaaggtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactcct gaagagc

Identification and Analysis of SAD2 Knockout/Knockdown Strains:

Construct D1283, derived from pSZ2282, was transformed into Strain J.Primary transformants were clonally purified and grown under standardlipid production conditions at pH 5. The resulting fatty acid profilesfrom representative clones arising from transformation with pSZ2282 intoStrain J are summarized in Table 39, below. D1283 transformantsaccumulated up to ˜42% C18:0 at the expense of C18:1, indicating thatSAD activity was significantly reduced in these strains.

TABLE 39 Fatty acid profiles of D1283 [pSZ2282] primary transformants,compared to the wild-type parental strain, Strain J. Strain S1920D1283-4 D1283-7 D1283-19 D1283-27 D1283-40 D1283-24 Fatty C12:0 0.040.05 0.05 0.07 0.06 0.04 0.05 Acid C14:0 1.31 0.92 1.07 1.01 1.08 1.030.96 Area C16:0 26.68 28.23 29.21 27.24 27.67 27.02 27.07 % C16:1 0.780.05 0.06 0.08 0.33 0.14 0.12 C17:0 0.11 0.12 0.15 0.10 0.10 0.12 0.13C18:0 3.15 41.98 40.94 34.20 26.26 23.18 22.82 C18:1 59.30 19.37 18.1726.87 34.77 38.74 39.38 C18:2 7.47 6.22 7.43 7.42 7.31 7.25 7.38  C18:3α0.57 0.93 1.03 0.75 0.71 0.72 0.51 C20:0 0.32 1.81 1.67 1.75 1.35 1.361.23 C20:1 0.00 0.10 0.00 0.12 0.00 0.12 0.11 C22:0 0.05 0.17 0.13 0.200.16 0.16 0.15 C24:0 0.00 0.00 0.00 0.10 0.00 0.00 0.00 sum C18 70.4968.5 67.57 69.24 69.05 69.83 70.09 saturates 31.66 73.28 73.23 64.6756.68 52.91 52.41 unsaturates 68.12 26.67 26.69 35.24 43.12 46.97 47.50

In Table 39, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text.

The fatty acid profiles of transformants D1283-4 and -7 were determinedto be stable after more than 30 generations of growth in the absence ofselection (growth on sucrose). The performance of selected strains inshake flask assays was then evaluated, and the fatty acid profiles andlipid titers are presented in Table 40, below. Strain X had the highestlevel of C18:0 (˜44%) and the best lipid titer (˜26%) relative to theStrain J parent, and so was selected for further fermentationdevelopment.

TABLE 40 Fatty acid profiles and lipid titers of SAD2knockout/knock-down derived from D1283 primary transformants, comparedto the wild-type parental strain, Strain J. Primary T342; D1283-4 T342;D1283-7 Strain Strain J S4490 S4491 S4492 S4493 S4494 K-4 Fatty C14:01.59 1.61 1.58 1.55 1.81 1.84 1.34 Acid C16:0 30.47 29.41 28.58 29.2428.77 29.09 28.47 Area C16:1 0.82 0.05 0.07 0.05 0.07 0.05 0.06 % C17:00.10 0.30 0.29 0.28 0.46 0.37 0.19 C18:0 3.58 42.85 41.86 43.38 39.9941.41 44.42 C18:1 56.96 13.52 15.55 13.49 13.57 12.98 15.64 C18:2 5.508.01 7.85 7.65 10.37 9.47 5.72  C18:3α 0.37 0.78 0.73 0.82 0.95 0.910.64 C20:0 0.22 2.06 2.11 2.11 1.98 1.98 2.32 C22:0 0.05 0.32 0.34 0.330.33 0.32 0.35 C24:0 0.03 0.43 0.42 0.44 0.49 0.49 0.37 lipid titer 10012.3 12.6 13.6 6.2 8.2 25.9 (% parent)

In Table 40, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text.

We optimized the performance of strain X in 7-L fermentations, and foundthat we could match the ˜44% C18:0 level obtained in shake flasks, withlipid productivities that were ˜45% of the wild-type parent. The fattyacid profiles and lipid titers of representative strain X fermentationsare summarized in Table 41, below. Fermentation of strain X underoptimal conditions yielded nearly 44% C18:0, which was similar to thestearate level that accumulated in shake flask assays. Strain K-4produced high C18:0 levels at both flask and 7-L scale and hadacceptable lipid productivity in 7-L fermentations; consequently thisstrain was selected as a base strain for additional modifications aimedat increasing C18:0 accumulation.

TABLE 41 Fatty acid profiles and lipid titers of strain X, compared to acontrol transgenic strain Strain Y. Strain Strain Y K-4 K-4 K-4Fermentation 110088F14 120489F5 120531F8 120580F1 Fatty Acid C14:0 1.471.18 1.15 1.27 Area % C16:0 25.66 28.68 28.38 28.35 C16:1 0.71 0.11 0.090.06 C18:0 3.16 41.63 42.40 43.67 C18:1 62.24 20.78 19.38 17.63 C18:25.90 5.06 5.38 5.58 C18:3α 0.16 0.24 0.25 0.25 C20:0 0.24 1.36 1.99 2.11C22:0 0.05 0.19 0.28 0.31 C24:0 0.05 0.34 0.29 0.31 sum C18 71.46 67.7167.41 67.13 saturates 30.63 73.38 74.49 76.02 unsaturates 69.01 26.1925.10 23.52 total lipid (g/L) 930 383 539 475

In Table 41, Stearate (C18:0) levels greater than the control arehighlighted with bold text. Strain Y contains S. cerevisiae SUC2,encoding sucrose invertase, integrated at the 6S locus, and has a fattyacid profile that is indistinguishable from the Strain J wild-typeparent.

Constructs Used for KASII Overexpression in Strain K-4:

DNA construct pSZ2734 was made to overexpress a codon-optimized P.moriformis KASII gene in Strain X. The neoR gene from transposon Tn5,conferring resistance to aminoglycoside antibiotics, was used as aselectable marker for transformation. The sequence of the transformingDNA is provided immediately below. Relevant restriction sites areindicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, XbaI,MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, BglII, AflII, HinDIII andSacI, respectively. BspQI sites delimit the 5′ and 3′ ends of thetransforming DNA. Underlined sequences at the 5′ and 3′ flanks of theconstruct represent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at the6S locus. Proceeding in the 5′ to 3′ direction, the C. reinhardtii TUB2promoter driving the expression of neoR (encoding aminoglycosidephosphotransferase activity, thereby permitting the strain to grow onG418) is indicated by lowercase, boxed text. The initiator ATG andterminator TGA for neoR are indicated by uppercase italics, while thecoding region is indicated with lowercase italics. The 3′ UTR of the C.vulgaris NR gene is indicated by small capitals, followed by a spacerregion indicated by lowercase text. The P. moriformis SAD2-2 promotersequence, indicated by boxed text, drives expression of thecodon-optimized P. moriformis KASII gene. The region encoding the KASIIplastid targeting sequence is indicated by uppercase italics. Thesequence that encodes the mature P. moriformis KASII polypeptide isindicated with bold, underlined, uppercase italics, while a 3×FLAGepitope encoding sequence is in bold italics. A second C. vulgaris NR 3′UTR is indicated by small capitals.

Nucleotide sequence of the transforming DNA from pSZ2734:(SEQ ID NO: 95) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggtgtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgcccagccgct

tatcaATGatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcac

GCAGACCGCCCACCAGCGCCCCCCCACCGAGGGCCACTGCTTCGGCGCCCGCCTGCCCACCGCCTCCC

TGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

Overexpression of KASII in Strain X:

Construct D1643 derived from pSZ2734 was transformed into Strain X asdescribed previously. Primary transformants were clonally purified andgrown under standard lipid production conditions at pH 5. The resultingfatty acid profiles from representative clones arising fromtransformation of Strain X with D1643 are summarized in Table 42, below.Overexpression of KASII in the SAD2 knockout/knock-down strain K-4background resulted in multiple strains accumulating over 50% C18:0 andwith substantially reduced levels of C16:0. We also observed that KASIIover-expressing lines had lower overall ratios of saturated tounsaturated fatty acids compared to Strain X.

TABLE 42 Fatty acid profiles of D1653 [pSZ2734] primary transformants,compared to the Strain X base strain and the wild-type parental strain,Strain J. D1653- D1653- D1653- D1653- D1653- D1653- D1653- D1653- D1653-D1653- D1653- D1653- Strain J X 89 10A 2B 5B 7A 75 90 9B 72 6B B2 66Fatty C12:0 0.04 0.06 0.27 0.13 0.20 0.19 0.24 0.13 0.12 0.27 0.16 0.180.25 0.22 Acid C14:0 1.44 1.06 1.55 1.65 1.79 1.67 1.70 1.53 1.50 1.741.57 1.64 1.48 1.56 Area C16:0 29.23 29.83 8.16 11.45 10.68 10.11 9.2711.14 11.08 9.40 9.78 9.95 8.12 8.65 % C16:1 0.88 0.10 0.04 0.00 0.000.00 0.00 0.04 0.04 0.00 0.04 0.00 0.05 0.06 C18:0 2.97 40.17 54.2553.87 53.61 53.46 53.32 53.32 53.15 52.43 52.20 51.23 50.52 50.02 C18:158.07 20.15 23.52 22.12 22.20 23.48 24.02 22.73 23.45 23.94 25.21 26.0728.00 28.29 C18:2 6.25 5.25 6.75 6.05 6.42 6.25 6.56 6.19 5.96 6.88 6.286.31 6.59 6.31  C18:3α 0.50 0.68 0.79 0.88 0.78 0.79 0.79 0.85 0.82 0.860.78 0.78 0.78 0.83 C20:0 0.22 1.88 3.21 2.81 3.01 2.91 3.02 2.86 2.773.21 2.74 2.80 2.87 2.80 C20:1 0.02 0.07 0.19 0.21 0.34 0.27 0.28 0.120.11 0.41 0.14 0.30 0.28 0.26 C22:0 0.05 0.26 0.41 0.34 0.40 0.37 0.370.36 0.35 0.42 0.36 0.37 0.36 0.37 C24:0 0.04 0.27 0.49 0.38 0.42 0.410.45 0.38 0.36 0.46 0.39 0.37 0.41 0.41 sum C18 67.78 66.24 85.31 82.9283.01 83.98 84.69 83.09 83.38 84.11 84.47 84.39 85.89 85.45 saturates33.97 73.52 68.34 70.63 70.11 69.12 68.37 69.72 69.33 67.93 67.20 66.5464.01 64.03 unsaturates 65.71 26.23 31.29 29.26 29.74 30.79 31.65 29.9330.38 32.09 32.45 33.46 35.70 35.75

In Table 42, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text. Palmitate (C16:0) levels lower thanstrain X or Strain J are highlighted with bold. For three strains theratio of saturated to unsaturated fatty acids is ≦2:1; these arehighlighted with bold, italicized text.

Stable lines were isolated from the primary transformants shown in Table42. The fatty acid profiles and lipid titers of shake flask cultures arepresented in Table 43, below. The strains accumulated up to 55% C18:0,with as low as 7% C16:0, with comparable lipid titers to the strain Xparent. The saturates:unsaturates ratios were substantially reducedcompared to strain X. Strains AU and AV were selected for evaluation in3-L high-density fermentations.

TABLE 43 Shake flask assays of strains derived from D1653, expressingKASII, driven by the PmSAD2-2 promoter, targeted to the 6S locus.PPrimary D1653-6B D1653-9B D1653-10A D1653-72 D1653-89 SStrain S1920JKK-4X SS5664 SAU SSBM BBBN BBBO BBBP BBBQ BBBR AAV BBBS Fatty C10:0 0.020.04 0.08 0.09 0.12 0.06 0.06 0.08 0.09 0.12 0.12 0.12 Acid C12:0 0.040.09 0.28 0.29 0.35 0.20 0.20 0.23 0.26 0.32 0.32 0.33 Area % C14:0 1.421.12 1.81 1.66 1.73 1.75 1.72 1.50 1.61 1.38 1.43 1.38 C16:0 25.59 28.569.39 8.61 8.44 9.98 10.11 8.26 8.95 6.81 7.21 6.63 C16:1 1.03 0.10 0.060.05 0.06 0.06 0.06 0.04 0.04 0.03 0.03 0.03 C18:0 2.60 40.13 47.6052.47 55.12 50.25 49.73 54.56 54.01 52.96 53.68 52.12 C18:1 62.08 20.7427.78 23.93 21.31 25.37 25.70 22.86 22.87 24.37 23.99 25.17 C18:2 6.165.83 7.98 7.52 7.72 7.55 7.64 7.20 7.24 8.11 7.83 8.04 C18:3α 0.40 0.891.21 1.22 1.49 1.17 1.07 1.20 1.29 1.28 1.24 1.31 C20:0 0.18 1.82 2.622.93 2.75 2.65 2.66 2.97 2.72 3.43 3.10 3.59 C20:1 0.04 0.13 0.37 0.360.39 0.34 0.34 0.35 0.34 0.48 0.41 0.47 C20:1 0.07 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 0.15 0.08 0.11 0.09 0.11 0.100.10 0.09 0.10 0.12 0.10 0.12 C22:0 0.02 0.20 0.28 0.30 0.24 0.29 0.280.30 0.27 0.32 0.29 0.35 C24:0 0.00 0.03 0.16 0.29 0.00 0.03 0.15 0.160.02 0.05 0.04 0.07 Sum C18 71.23 67.58 84.57 85.13 85.63 84.34 84.1385.81 85.40 86.71 86.73 86.63 Saturates 29.86 71.97

68.74

68.05 67.90

Unsats 69.91 27.76

31.07

31.73 31.87

In Table 43, strain X is the parent strain; Strain J is the wild-typebase strain. Stearate (C18:0) levels at least two-fold higher than inthe wild-type strain are highlighted in bold. Palmitate levels that areless than in Strain J and strain X are highlighted bold. Bold italicsindicate that the saturates:unsaturates ratio is ≦2:1.

The fatty acid profiles and performance metrics of strains AU and AV aredetailed in Table 44, below. The fatty acid profile of the parent strainstrain K-4, grown under the same fermentation conditions, is presentedfor comparison. The strains that over-express KASII accumulate about 11%more C18:0 than the strain K-4 parent. C16:0 is reduced to 7-9%, andlevels of unsaturated fatty acids increase by 4-5%. The lipid titers ofstrains AU and AV were comparable to strain X, indicating that KASIIover-expression did not have deleterious effects on lipid production.

TABLE 44 End point fatty acid profiles of biomass from strains X, AU andAV fermentations. Strain X AU AV Fermentation 120580F1 130097F3 130098F4pH 5 5 5 C14:0 1.27 1.50 1.35 C16:0 28.35 8.88 7.33 C16:1 0.06 0.02 0.03C18:0 43.67 56.88 57.24 C18:1 17.63 21.57 21.66 C18:2 5.58 6.06 6.94C18:3α 0.25 0.29 0.22 C20:0 2.11 3.28 3.46 C22:0 0.31 0.40 0.40 C24:00.31 0.37 0.40 sum C18 67.13 84.80 86.06 saturates 76.02 71.31 70.18unsaturates 23.52 27.94 28.85 total lipid (g/L) 475 529 418

The fermentations were cultured for 6 days using a fed-batch process.The Strain X fatty acid profile from fermentation 120580F1 was presentedin Table 41, and is shown again in Table 44 for comparison with StrainsAU and AV. All fermentations were carried out at 32° C., pH 5, 30%dissolved oxygen (DO), 300 mM nitrogen [N], and 557.5 μM iron. The sugarsource was 70% sucrose (S70). Stearate (C18:0) levels higher than in thewild-type strain are indicated with bold. Palmitate (C16:0) levels thatare less than in the wild-type are highlighted bold.

Lab scale oils were prepared from biomass derived from the shake flasksand fermentations described above. The TAG compositions of these oilswere determined by LC/MS. SOS is the major TAG species in both StrainsAU and AV, ranging from 33-35% in the biomass from shake flasks, andreaching 37% in the high-density fermentation biomass. The majorpalmitate-containing TAGs are substantially reduced, and trisaturatelevels are less than half of those observed in Strain X oils. Theseresults demonstrate that KASII over-expression in a high-stearatebackground significantly improves SOS accumulation, and reduces theaccumulation of trisaturated TAGs.

Constructs Used for FATA-1 Disruption, KASII Over-Expression and FAD2RNAi in Strain J:

A DNA construct, pSZ2419, was made to simultaneously disrupt the FATA-1allele, over-express P. moriformis KASII and express a FAD2 hairpinconstruct in Strain J. A version of the S. cerevisiae SUC2 gene,encoding sucrose invertase, which was codon-optimized for expression inP. moriformis, was utilized as a selectable marker for transformation.The sequence of the transforming DNA is provided immediately below.Relevant restriction sites are indicated in lowercase, bold, and arefrom 5′-3′ BspQI, KpnI, AscI, MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI,AscI, ClaI, BglII, AflII, HinDIII, SacI, SpeI, and XhoI, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA.Underlined sequences at the 5′ and 3′ flanks of the construct representgenomic DNA from P. moriformis that enable targeted integration of thetransforming DNA via homologous recombination at the FATA-1 locus.Proceeding in the 5′ to 3′ direction, the C. reinhardtii TUB2 promoterdriving the expression of the S. cerevisiae SUC2 gene (encoding sucrosehydrolyzing activity, thereby permitting the strain to grow on sucrose)is indicated by lowercase, boxed text. The initiator ATG and terminatorTGA for SUC2 are indicated by uppercase italics, while the coding regionis indicated with lowercase italics. The 3′ UTR of the C. vulgarisnitrate reductase (NR) gene is indicated by small capitals, followed bya spacer region indicated by lowercase text. The P. moriformis AMT3promoter, indicated by lowercase boxed text, drives expression of the P.moriformis KASII gene. The region encoding the plastid targeting peptidefrom Chlorella protothecoides SAD1 is indicated by uppercase italics.The sequence that encodes the mature P. moriformis KASII polypeptide isindicated with bold, underlined, uppercase italics, while a 3×FLAGepitope encoding sequence is in bold italics. A second C. vulgaris NR 3′UTR is indicated by small capitals. A second C. reinhardtii TUB2promoter sequence, indicated by lowercase boxed text, drives expressionof the P. moriformis FAD2 hairpin A sequence. The sense and antisensestrands are indicated with uppercase, bold italics, and are separated bythe FAD2 intron and the first 10 bases of the FAD2 second exon(uppercase italics). A third C. vulgaris NR 3′ UTR is indicated by smallcapitals, followed by a second spacer region that is indicated bylowercase text.

Nucleotide sequence of the transforming DNA from pSZ2419:(SEQ ID NO: 96) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtcagcaccgccagccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactggcgtctcttccgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccgcagcggccgctgcccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacccgcccaagagttttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtggaggtccggggctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggccagaaacactgtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactctttctcctcctccttcccgttcacgcagcattcggggtacc

ggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgg

TTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCA

GTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGA

GCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagctgtattgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaagacagggtggttggctggatggggaaacgctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacacacacaacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctccctgccgcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggccccttgtgcgcagtgctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggccaagcgggctgctgggcgcgccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccacgcgcttgctgctttgggaagagaagggggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttgggt gaagagc

Identification and Analysis of FATA-1 Knockout, KASII Over-Expressionand FAD2 RNAi Strains:

Construct D1358, derived from pSZ2419, was transformed into Strain J asdescribed previously. Primary transformants were clonally purified andgrown under standard lipid production conditions at pH 5. The resultingfatty acid profiles from representative clones arising fromtransformation of Strain J with D1358 are summarized in Table 45, below.The P. moriformis AMTS promoter is repressed at pH 5 so the observedphenotypes did not reflect over-expression of P. moriformis KASII.Nevertheless, we observed that multiple strains had substantiallyreduced levels of C16:0 and 10-15% increases in C18:1, suggesting thatthe construct had disrupted the FATA-1 target gene, increasing theamount of palmitoyl-ACP available for extension by endogenous KASII. Oneline, D1358-13, was selected for further analysis. D1358-13 accumulated˜17% C16:0, ˜75% C18:1 and less than 2% C18:2, indicating that we hadsuccessfully integrated at FATA-1 and downregulated activity of the FAD2Δ¹²-desaturase in this strain.

TABLE 45 Fatty acid profiles of D1358 [pSZ2419] primary transformants,compared to the wild-type parental strain, Strain J. D1358- D1358-D1358- D1358- D1358- D1358- D1358- D1358- D1358- D1358- Strain J 13 1911 9 30 28 6 8 10 3 Fatty C12:0 0.05 0.08 0.06 0.08 0.06 0.07 0.07 0.090.07 0.08 0.10 Acid C14:0 1.32 0.79 0.83 0.85 0.87 0.84 0.91 0.86 0.890.92 0.60 Area % C16:0 26.66 17.43 18.84 20.03 16.27 18.4 19.1 18.1815.6 16.42 11.24 C16:1 0.84 0.74 0.79 0.97 0.60 0.77 1.17 0.75 0.56 0.610.57 C18:0 3.10 2.87 2.97 2.36 3.20 2.67 2.10 2.82 3.22 3.19 2.30 C18:159.07 74.78 69.54 68.78 71.48 69.55 69.02 68.93 70.44 69.64 75.27 C18:27.39 1.97 5.47 5.61 6.22 6.31 6.42 6.8 7.68 7.78 8.51 C18:3α 0.55 0.230.59 0.51 0.26 0.39 0.46 0.38 0.24 0.27 0.24 C20:0 0.24 0.22 0.20 0.130.32 0.20 0.03 0.20 0.33 0.31 0.22 C20:1 0.11 0.40 0.29 0.37 0.23 0.330.33 0.39 0.36 0.27 0.40 C22:0 0.11 0.09 0.08 0.07 0.09 0.08 0.08 0.080.09 0.11 0.11 sum C18 70.11 79.85 78.57 77.26 81.16 78.92 78.00 78.9381.58 80.88 86.32 saturates 31.48 21.48 22.98 23.52 20.81 22.26 22.2922.23 20.20 21.03 14.57 unsaturates 67.96 78.12 76.68 76.24 78.79 77.3577.4 77.25 79.28 78.57 84.99

In Table 45, Oleate (C18:1) levels greater than the wild-type level arehighlighted with bold text. Palmitate (C16:0) levels less than thewild-type are highlighted with bold text. Levels of linoleate (C18:2)reduced by 1% or more compared to the Strain J parent are highlightedwith bold text.

The fatty acid profiles of strains derived from transformant D1358-13were determined to be stable after more than 60 generations of growth inthe absence of selection (growth on sucrose). The performance ofselected strains in shake flask assays was then evaluated, and the fattyacid profiles and lipid titers are presented in Table 46, below. Flaskexperiments were performed at pH 7, enabling activation of the PmAMT3promoter driving expression of the KASII transgene. The combination ofKASII over-expression and FATA-1 knockout leads to further reductions inpalmitate levels and enhanced oleate accumulation compared to thephenotypes observed at pH 5 (Table 45). With more than 82% C18:1, lessthan 11% C16:0, less than 2% C18:2 and ˜83% of the wild-type lipidtiter, Strain AAStrain AA was determined to be the most appropriatestrain from this set to serve as a host strain for subsequentmodifications to elevate stearate levels. DNA blot analysis showed thatStrain AA has a simple insertion of construct D1358 [pSZ2419] at theFATA-1 locus.

TABLE 46 Fatty acid profiles and lipid titers of FATA-1 knockout, KASIIover-expressing, FAD2 RNAi lines derived from D1358-13 primarytransformants, compared to the wild-type parental strain, Strain J.Primary T389; D1358-13 Strain J AA AB AC AD AE AF AG AH AI AJ AK AL AMFatty C12:0  0.05  0.08  0.09  0.11  0.19  0.11  0.14  0.10  0.12  0.08 0.11  0.09  0.20  0.20 Acid C14:0  1.34  0.96  0.98  1.03  1.04  0.96 1.02  0.98  1.03  0.98  1.01  1.00  1.03  1.02 Area % C16:0  29.6910.72 10.47  8.90  6.99  9.53  9.27 10.13  8.99  10.76  9.58 10.00  6.64 6.38 C16:1  0.88  0.42  0.39  0.31  0.29  0.39  0.37  0.41  0.32  0.40 0.35  0.35  0.27  0.27 C18:0  2.78  2.92  3.00  3.16  2.71  2.88  2.85 2.91  3.21  3.03  3.10  3.20  2.77  2.71 C18:1  58.45 82.08 82.24 83.6685.49 83.28 83.38 82.57 83.51  82.12 83.10 82.63 85.88 86.13 C18:2  5.83 1.89  1.88  1.80  2.01  1.83  1.89  1.89  1.77  1.73  1.75  1.76  1.94 1.96 C18:3α  0.42  0.23  0.23  0.25  0.35  0.27  0.29  0.27  0.25  0.22 0.24  0.23  0.34  0.36 C20:0  0.17  0.15  0.16  0.17  0.15  0.15  0.16 0.16  0.17  0.14  0.16  0.16  0.15  0.15 C20:1  0.05  0.23  0.24  0.27 0.36  0.28  0.29  0.26  0.27  0.21  0.25  0.24  0.38  0.39 sum C18 67.48 87.12 87.35 88.87 90.56 88.26 88.41 87.64 88.74  87.10 88.1987.82 90.93 91.16 saturates  34.03 14.83 14.70 13.37 11.08 13.63 13.4414.28 13.52  14.99 13.96 14.45 10.79 10.46 unsaturates  65.63 84.8584.98 86.29 88.50 86.05 86.22 85.40 86.12  84.68 85.69 85.21 88.81 89.11lipid titer 100.0  82.8  81.1  72.8  54.4  68.3  63.7  70.6  72.2 106.9  76.5  77.5  56.7  54.6  (% parent)

In Table 46, Stearate (C18:1) levels greater than the wild-type levelare highlighted with bold text. Palmitate (C16:0) levels lower than thewild-type are highlighted with bold text. Linoleate (C18:2) levels thatare lower than the wild-type are indicated with bold text.

Constructs Used for SAD2 Knockout/RNAi in Strain AA:

Two DNA constructs, pSZ2283 and pSZ2697, were made to simultaneouslydisrupt the SAD2-1 allele and express a SAD2 hairpin construct in StrainAA. In each construct, the neoR gene from transposon Tn5, conferringresistance to aminoglycoside antibiotics, was used as a selectablemarker for transformation. The sequence of the transforming DNA derivedfrom pSZ2283 is provided immediately below. Relevant restriction sitesare indicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, XbaI,MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI,respectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Underlined sequences at the 5′ and 3′ flanks of the constructrepresent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the Chlamydomonasreinhardtii TUB2 promoter driving the expression of neoR (encodingaminoglycoside phosphotransferase activity, thereby permitting thestrain to grow on G418) is indicated by lowercase, boxed text. Theinitiator ATG and terminator TGA for neoR are indicated by uppercaseitalics, while the coding region is indicated with lowercase italics.The 3′ UTR of the C. vulgaris NR gene is indicated by small capitals,followed by a spacer region indicated by lowercase text. A second C.reinhardtii TUB2 promoter sequence, indicated by lowercase boxed text,drives expression of the SAD2 hairpin C sequence. The sense andantisense strands are indicated with uppercase, bold italics, and areseparated by the P. moriformis FAD2 intron and the first 10 bases of theFAD2 second exon (uppercase italics). A second C. vulgaris NR 3′ UTR isindicated by small capitals.

Nucleotide sequence of the transforming DNA from pSZ2283:(SEQ ID NO: 97) gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaacaacccttattt

ggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgg

GTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagctggagctccagccacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatgatgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaaggtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactcctgaag agc

The sequence of the transforming DNA derived from pSZ2697 is providedimmediately below. Relevant restriction sites are indicated inlowercase, bold, and are from 5′-3′ NsiI, SpeI, BamHI, HinDIII, SacII,EcoRV, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, EcoRI and XbaI,respectively. Underlined sequences at the 5′ and 3′ flanks of theconstruct represent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the SAD2 hairpin Csense and antisense strands are indicated with uppercase, bold italics,and are separated by the P. moriformis FAD2 intron and the first 10bases of the FAD2 second exon (uppercase italics). The 3′ UTR of the C.vulgaris NR gene is indicated by small capitals. The Chlorellasorokiniana Glutamate Dehydrogenase (GDH) promoter, driving theexpression of neoR (encoding aminoglyco side phosphotransferaseactivity, thereby permitting the strain to grow on G418) is indicated bylowercase, boxed text. The initiator ATG and terminator TGA for neoR areindicated by uppercase italics, while the coding region is indicatedwith lowercase italics. A second C. vulgaris NR 3′ UTR is indicated bysmall capitals, followed by a spacer region indicated by lowercase text.

Nucleotide sequence of the transforming DNA from pSZ2697:(SEQ ID NO: 98) atgcatgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacggaaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgacacgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagcagttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcgcaggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcggaacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcaggcatcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtggcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactctcccgactagt

GACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAA

tagaatatcaATGatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctagggatatcgaattccgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaacaacccttatttggggttcgtctacacctccttccaggagcgcgccaccaagtatctaga

Identification and Analysis of SAD2 Knockout/Knockdown Strains in theStrain AA Background:

Constructs D1639, derived from pSZ2697, and D1682, derived from pSZ2283,were transformed into Strain AA as described previously. Primarytransformants were clonally purified and grown under standard lipidproduction conditions at pH 7. The resulting fatty acid profiles fromrepresentative clones arising from transformation are summarized inTable 47, below. D1639 transformants accumulated up to 56% C18:0, andD1682 transformants accumulated a maximum of about 35% C18:0. Most ofthe increases in stearate came at the expense of C18:1, indicating thatSAD activity was significantly reduced by the SAD2 knockout/RNAiconstructs in these strains. C16:0 levels varied from 6% to 14%; C18:2ranged from 2-5%. Most strains maintained the low C16:0 and C18:2phenotypes of the Strain AA parent. These fatty acid profilesdemonstrate that down-regulating SAD2 expression using knockout/RNAiconstructs, in a background with disrupted FATA-1, KASII over-expressionand FAD2 RNAi, produces strains with high C18:0, low C16:0 and low C18:2phenotypes. These strains will be useful for production of highstability, high stearate, high oleic oils, and oils which have high SOScontent.

TABLE 47 Fatty acid profiles of D1639 [pSZ2697] and D1682 [pSZ2283]primary transformants, compared to the wild-type strain, Strain J, andthe Strain AA parental base strain. D1682- D1682- D1682- D1682- D1639-D1639- D1639- D1639- Strain J AA 4 17 7 6 2 5 10 19 Fatty C12:0 0.040.11 0.14 0.10 0.32 0.31 0.00 0.19 0.17 0.00 Acid C14:0 1.29 0.98 1.030.94 1.11 1.15 1.64 1.39 1.61 1.02 Area % C16:0 27.50 7.75 8.68 10.415.70 5.96 7.54 9.90 14.39 12.02 C16:1 0.71 0.30 0.06 0.07 0.07 0.10 0.000.00 0.00 0.00 C18:0 3.28 3.60 35.46 29.92 24.66 22.30 55.96 53.38 43.4637.30 C18:1 57.80 84.14 48.39 52.49 61.04 63.60 23.70 26.79 32.93 42.81C18:2 7.90 2.09 2.37 2.36 3.03 2.88 5.09 3.50 3.22 2.79 C18:3α 0.57 0.320.50 0.65 0.66 0.58 1.59 0.98 1.01 0.85 C20:0 0.28 0.23 2.07 1.87 1.751.51 3.04 2.73 2.29 2.22 C20:1 0.18 0.35 0.54 0.49 0.78 0.83 0.37 0.330.30 0.40 C22:0 0.06 0.02 0.27 0.27 0.23 0.20 0.43 0.36 0.29 0.29 C24:00.09 0.02 0.33 0.26 0.34 0.26 0.64 0.45 0.32 0.31 sum C18 69.55 90.1486.72 85.42 89.39 89.36 86.34 84.65 80.62 83.75 saturates 32.54 12.7047.98 43.77 34.11 31.69 69.25 68.40 62.53 53.16 unsaturates 67.16 8 7.2151.86 56.06 65.58 67.99 30.75 31.60 37.46 46.85

In Table 47, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text. Oleate (C18:1) levels that are higherthan in the wild-type are indicated with bold text. Palmitate (C16:0)levels less than the wild-type level are highlighted with bold. Reducedlevels of linoleate (C18:2) compared to the wild-type are highlightedwith bold text.

Stable lines were isolated from a number of D1639 and D1682transformants. Shake flask assays were carried out to evaluate theperformance of four lines derived from D1639-5. Fatty acid profiles andrelative lipid titers from the biomass are shown in Table 48, below.

TABLE 48 Shake flask assays of strains derived from D1639-5, expressingSAD2hpC, driven by the CrTUB2 promoter, targeted to the SAD2-1 locus.Primary T530; D1639-5 Strain J AA AW AX AY BL Fatty C10:0 0.01 0.00 0.070.08 0.05 0.04 Acid C12:0 0.02 0.11 0.19 0.22 0.25 0.23 Area % C14:01.52 1.10 1.35 1.32 1.30 1.43 C16:0 31.61 9.59 9.28 8.44 7.74 9.46 C16:11.04 0.34 0.03 0.02 0.01 0.01 C17:0 0.10 0.11 0.10 0.10 0.10 0.09 C18:02.98 4.36 53.01 53.52 55.32 52.09 C18:1 54.81 80.84 27.26 27.52 27.4228.06 C18:2 6.88 2.42 3.55 3.52 2.38 3.45 C18:3α 0.53 0.33 0.97 1.030.82 1.06 C20:0 0.26 0.31 2.88 2.94 3.15 2.72 C20:1 0.05 0.34 0.38 0.380.40 0.37 C22:0 0.03 0.06 0.36 0.37 0.39 0.35 C24:0 0.07 0.08 0.53 0.540.53 0.60 sum C18 65.19 87.95 84.79 85.58 85.94 84.66 saturates 36.5915.70 67.76 67.52 68.82 66.99 unsaturates 63.30 84.26 32.19 32.46 31.0232.95 % wild-type 100.0 70.3 34.8 33.7 31.4 35.3 lipid titer

In Table 48, Strain AA is the parent strain; Strain J is the wild-typebase strain. Stearate (C18:0) levels higher than in the wild-type strainare indicated with bold. Bold text indicates the increased level ofoleate (C18:1) in Strain AA compared to the wild-type. Palmitate (C16:0)levels that are less than in the wild-type are highlighted bold.Linoleate (C18:2) levels that are less than in the wild-type areindicated with bold.

Lab scale oils were prepared from biomass collected from the Strains AW,AX and AY shake flasks. The TAG compositions of these oils weredetermined by LC/MS, and are shown in FIG. 21. SOS accumulation rangedfrom 42-47% in these strains. POS was the next most abundant TAG, at16-17%. Linoleate-containing TAGs were reduced by more than 50% comparedto the Strain AU and AV oils, described above. Strains AW, AX, and AYoils contained 12-13% trisaturated TAGs (S—S—S), similar to the amountsthat accumulated in the Strains AU and AX oils. Modulation of SADactivity during oil production to prevent overproduction of saturatedfatty acids may help to reduce accumulation of trisaturates.

Example 49 Properties of Methyl Oleate from High Oleic Microalgal Oils

Esterified oils high in methyl oleate are useful in a variety ofapplications such as cleaning and lubrication of machinery. For some ofthese applications, high thermal stability is desired. Thermal stabilitytesting was performed on methylated oil prepared from high-oleic andhigh-stability-high oleic triglyceride oils prepared fromheterotrophically grown oleaginous microalgae as described above. Theoils were bleached and deodorized prior to methylation. Commerciallyavailable soya methyl ester was used as a control.

High Oleic (HO) oil was prepared from a high oil-yielding strain ofPrototheca moriformis transformed with a plasmid that can be describedas FatA1_Btub:inv:nmamt03-CwTE2:nr_FatA1. This plasmid was designed tohomologously recombine in the FATA1 chromosomal site, thus ablating aFATA acyl-ACP thioesterase chromosomal allele, while expressing anexogenous acyl-ACP thioesterase from Cuphea. wrightii (CwTE2, SEQ ID NO:11) under control of the pH-regulatable amt3 promoter. The CwTE2 genecan be downregulated by cultivation at pH 5 during oil production tofurther elevate oleate production. Sucrose invertase was also expressedas a selection marker and to allow for cultivation of the strain onsucrose as a sole carbon source. The 3′ UTR sequences are from theChlorella vulgaris nitrate reductase gene. The resulting HO strain isdenoted Stain Q. The fatty acid profile of the oil produced by Strain Qis listed below in Table 49.

TABLE 49 Fatty acid profile of high oleic oil from Strain Q. Fatty AcidArea % C10 0.01 C12:0 0.03 C14:0 0.43 C15:0 0.03 C16:0 7.27 C16:1 iso0.81 C16:1 0.689 C17:0 0.06 C18:0 1.198 C18:1 80.15 C18:1 iso 0.08 C18:28.38 C18:3 ALPHA 0.25 C20:0 0.02 C20:1 0.38 C22:0 0.04 C24:0 0.03

A high-stability-high-oleic oil (HSAO) was also prepared from a highoil-yielding strain of Prototheca moriformis transformed with a plasmidthat can be described as FADc5′_Btub:inv:nr::btub-CpSAD_CtOTE:nr_FADc3′(pSZ1501, SEQ ID NO:111). The resulting strain (Strain R) expressessucrose invertase as a selectable marker and to allow for cultivation onsucrose as a sole carbon source. In addition, a FAD allele (encodingfatty acid desaturase responsible for the conversion of oleate tolinoleate) is disrupted and an oleate-specific acyl-ACP thioesterase(Carthamus tinctorius OTE, see example 5) fused to the transit peptidefrom the SAD gene of Chlorella protothecoides is expressed under controlof the beta tubulin promoter. The 3′ UTR sequences are from theChlorella vulgaris nitrate reductase gene. The fatty acid profile of theoil produced by Strain R after heterotrophic cultivation is listed belowin Table 50. The fatty acid profile has greater than 85% oleate yetalmost none of the major polyunsaturates, linoleic and linolenic acids.

TABLE 50 Fatty acid profile of high oleic oil from Strain R. Fatty AcidArea % C10 0.02 C12:0 0.07 C14:0 0.09 C15:0 0.05 C16:0 7.28 C16:1 0.70C17:0 0.08 C18:0 2.15 C18:1 86.32 C20:0 0.30 C20:1 0.46 C22:0 0.08 C23:00.01 C24:0 0.06

The HO and HSAO oils were methylated by known biodiesel productiontechniques to make methyl-HO and methyl-HSAO esters. These methyl esterswhere then subjection to thermal testing according to the followingprocedure:

-   -   1. Prepare equipment as shown in FIG. 1.    -   2. Add 1 litre of water to test vessel and bring to an active        boil on the hotplate.    -   3. To each test product add 50 ppm Cobalt (0.083 g of 6% Cobalt        Napthenate in 100.0 gram sample) and mix thoroughly.    -   4. Weigh out, in a watch glass, 7.0 g of 100% cotton gauze, (#50        Cheese Cloth).    -   5. Evenly distribute 14.0 g of test product, as prepared in step        3, onto the gauze.    -   6. Place thermocouple (thermometer) through the center of #15        stopper. Wrap cotton around thermocouple.    -   7. Place wrapped cotton into 24 mesh wire frame cylinder so that        it occupies the upper 4½ h inches.    -   8. Position cylinder with wrapped gauze into the 1 L tall form        beaker. Secure the beaker in the boiling water and begin        recording the temperature increase with time.    -   9. Continue monitoring the temperature for 2 hours or until a 10        degree temperature drop in observed.    -   10. Plot temperature vs time on a graph.    -   11. Any sample which shows a temperature exceeding 100        degrees C. in 1 hour or 200 degrees C. in 2 hours should be        regarded as a dangerous oxidation risk or one that is likely to        spontaneously combust.

Results: The HO and HSAO methyl ester did not exhibit auto-oxidation asevidenced by a temperature rise. The control soya methyl ester sampledid exhibit the potential for auto-oxidation. The time-temperatureprofiles are shown in FIG. 18.

In addition, methylated fatty acid from oil produced by Strain Q wasfound to have the following characteristics:

Flash Point (ASTM D93) of 182° C.

Non-VOC

Kauri Butanol value (ASTM D1133) of 53.5

Viscosity at 40° C. (ASTM D445) of 4.57 mm2/s

Acid Number (ASTM D664) of 0.17 mg KOH/g

Boiling range distribution (ASTM D2887) 325-362° C.

Example 50 Further Properties of High Oleic (HO) andHigh-Stability-High-Oleic (HSAO) Microalgal Oils

The high oleic oil and the high-stability high-oleic algal oils can havethe properties shown in FIG. 19 or these values±20% for the measuredparameters.

In one experiment, HSAO microalgal oil showed 512 hour stabilitymeasured by OSI at 110° C. (estimated using 130° C. data) withantioxidants of 0.5% phenyl-alpha-naphthylamine (PANA) and 500 ppmascorbyl palmitate (AP).

Example 51 Production of Low Saturate Oil by Conversion of Palmitic toPalmitoleate

As described in the examples above, genetic manipulation of microalgaecan decrease saturated fat levels, especially by increasing theproduction of oleic acid. However, in some cases, the acyl-ACPthioesterases expressed in the oleaginous cell liberate more thandesirable amounts of palmitate. Here, we describe methods for convertingpalmitate (16:0) to palmitoleate (16:1) by overexpressing apalmitoyl-ACP desaturase (PAD) gene. The PAD gene can be obtained fromnatural sources such as Macfadyena unguis (Cat's claw), Macadamiaintegrifolia (Macadamia nut), Hippophae rhamnoides (sea buckthorn), orby creating a PAD via mutation of a stearoyl-ACP desaturase to have 16:1activity. The Macfadyena unguis desaturase is denoted (MuPAD).

A high-oil-producing strain of Prototheca moriformis (Strain Z) isbiolistically transformed with plasmid DNA constructs with a PAD gene.For example, one of the high oleic strains described in the Examples 6,36, or 49 can further comprise an exogenous PAD gene. The constructscomprises sucrose invertase as a selectable marker and either the MuPADor a SAD gene (e.g., Olea europaea stearoyl-ACP desaturase, GenBankAccession No. AAB67840.1) having the L118W mutation to shiftsubstrate-specificity toward palmitate. See Cahoon, et al., PlantPhysoil (1998) 117:593-598. Both the amt3 and beta tubulin (Btub)promoters are used. In addition, the native transit peptide of a plantPAD gene can be swapped with one known to be effective in microalgae(e.g., the transit peptide from the Chlorella vularis SAD gene).

The PAD gene can be expressed in a variety of strains including thosewith a FATA knockout or knockdown and/or a KASII knockin to producehigh-oleic oil. Optionally, these strains can also producehigh-stability (low polyunsaturate) oil by virtue of a FAD (delta 12fatty acid desaturase) knockout, knockdown, or by placing FAD expressionunder control of a regulatable promoter and producing oil underconditions that downregulate FAD. In addition, useful base strains forthe introduction of PAD gene activities might also include strainspossessing KASII knockouts, and FATA Knockins, whereby levels of C16:0palmitate are elevated.

As a result, lower levels of palmitic acid are found in the fatty acidprofile of the microalgal oil as this is converted into cis-palmitoleicand cis-vaccenic acids. In some cases the total area percent ofsaturated fatty acids is less than equal to 3.5%, 3% or 2.5%.

Constructs for over expression of Macfadyena unguis C16:0 desaturase(MuPAD) follow:

1) pSZ3142: 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ31426S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of Prototheca moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the MuPAD are indicated byuppercase, bold italics, while the remainder of the coding region isindicated by bold italics. The Chlorella protothecoides S106stearoyl-ACP desaturase transit peptide is located between initiator ATGand the Asc I site. The C. vulgaris nitrate reductase 3′ UTR is againindicated by lowercase underlined text followed by the 6S genomic regionindicated by bold, lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3142:(SEQ ID NO: 99) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgccc

gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagaccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggt

ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcg ggcgcgccgccaccctgcgctccggcctgcgcgacgtggagaccgtgaagaagaccttctcccccgcccgcgaggtgcacgtgcaggtgacccactccatggccccccagaagatcgagatcttcaaggccatggaggactgggccgagaacaacatcctggtgcacctgaagaacgtggagaagtgcccccagccccaggacttcctgcccgaccccgcctccgacgagttccacgaccagatcaaggagctgcgcgagcgcgccaaggagatccccgacgactacttcgtggtgctggtgggcgacatgatcaccgaggaggccctgcccacctaccagaccatgctgaacacctgggacggcgtgcgcgacgagaccggcgcctcccccacctcctgggccatctggacccgcgcctggaccgccgaggagaaccgccacggcgaccccctgaacaagtacctgtacctgtccggccgcgtggacatgaagcagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccgagaactccccctacctgggcttcatctacacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgcctggcccgcgaccacggcgacttcaagctggcccagatctgcggcaccatcgcctccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccccgacggcaccgtgctggccttcggcgacatgatgaagaagaagatctccatgcccgaccacttcatgtacgacggccgcgacgacaacctgttcgaccacttctcctccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagcacctggtgggccgctggaaggtggagaagctgaccggcctgtccgccgagggccagaaggcccaggactacgtgtgcggcctgcccccccgcatccgccgcctggaggagcgcgcccagatccgcgccaagcaggccccccgcctgcccttctcctggatctacgaccgcgaggtgcagctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGAatcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctc ttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc  

2) pSZ3145: 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ31456S::CrTUB2:ScSUC2:CvNR::PmAMT3: MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQIsites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of Prototheca moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the MuPAD are indicated byuppercase, bold italics, while the remainder of the coding region isindicated by bold italics. The C. vulgaris nitrate reductase 3′ UTR isagain indicated by lowercase underlined text followed by the 6S genomicregion indicated by bold, lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3145:(SEQ ID NO: 100) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgccc

gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggt

gccccccgtggtgtccctgcgctcccccaagctgtccgtggccgccaccctgcgctccggcctgcgcgacgtggagaccgtgaagaagaccttctcccccgcccgcgaggtgcacgtgcaggtgacccactccatggccccccagaagatcgagatcttcaaggccatggaggactgggccgagaacaacatcctggtgcacctgaagaacgtggagaagtgcccccagccccaggacttcctgcccgaccccgcctccgacgagttccacgaccagatcaaggagctgcgcgagcgcgccaaggagatccccgacgactacttcgtggtgctggtgggcgacatgatcaccgaggaggccctgcccacctaccagaccatgctgaacacctgggacggcgtgcgcgacgagaccggcgcctcccccacctcctgggccatctggacccgcgcctggaccgccgaggagaaccgccacggcgaccccctgaacaagtacctgtacctgtccggccgcgtggacatgaagcagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccgagaactccccctacctgggcttcatctacacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgcctggcccgcgaccacggcgacttcaagctggcccagatctgcggcaccatcgcctccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccccgacggcaccgtgctggccttcggcgacatgatgaagaagaagatctccatgcccgaccacttcatgtacgacggccgcgacgacaacctgttcgaccacttctcctccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagcacctggtgggccgctggaaggtggagaagctgaccggcctgtccgccgagggccagaaggcccaggactacgtgtgcggcctgcccccccgcatccgccgcctggaggagcgcgcccagatccgcgccaagcaggccccccgcctgcccttctcctggatctacgaccgcgaggtgcagctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGA atcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

3) pSZ3137: 6S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ31376S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by C. reinhardtiitubulin promoter, indicated by boxed italics text. The Initiator ATG andterminator TGA codons of the MuPAD are indicated by uppercase, bolditalics, while the remainder of the coding region is indicated by bolditalics. The Chlorella protothecoides S106 stearoyl-ACP desaturasetransit peptide is located between initiator ATG and the Asc I site. TheC. vulgaris nitrate reductase 3′ UTR is again indicated by lowercaseunderlined text followed by the 6S genomic region indicated by bold,lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3137:(SEQ ID NO: 101) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgccc

gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagaccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggt

ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcg ggcgcgccgccaccctgcgctccggcctgcgcgacgtggagaccgtgaagaagaccttctcccccgcccgcgaggtgcacgtgcaggtgacccactccatggccccccagaagatcgagatcttcaaggccatggaggactgggccgagaacaacatcctggtgcacctgaagaacgtggagaagtgcccccagccccaggacttcctgcccgaccccgcctccgacgagttccacgaccagatcaaggagctgcgcgagcgcgccaaggagatccccgacgactacttcgtggtgctggtgggcgacatgatcaccgaggaggccctgcccacctaccagaccatgctgaacacctgggacggcgtgcgcgacgagaccggcgcctcccccacctcctgggccatctggacccgcgcctggaccgccgaggagaaccgccacggcgaccccctgaacaagtacctgtacctgtccggccgcgtggacatgaagcagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccgagaactccccctacctgggcttcatctacacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgcctggcccgcgaccacggcgacttcaagctggcccagatctgcggcaccatcgcctccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccccgacggcaccgtgctggccttcggcgacatgatgaagaagaagatctccatgcccgaccacttcatgtacgacggccgcgacgacaacctgttcgaccacttctcctccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagcacctggtgggccgctggaaggtggagaagctgaccggcctgtccgccgagggccagaaggcccaggactacgtgtgcggcctgcccccccgcatccgccgcctggaggagcgcgcccagatccgcgccaagcaggccccccgcctgcccttctcctggatctacgaccgcgaggtgcagctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGAatcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctc ttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc  

Example 52 Myristate Rich Oil Produced by Overexpressing a Cupheapalustris Thioesterase

Here, we demonstrate that over expression of a Cuphea palustristhioesterase (Cpal FATB2, accession AAC49180) in UTEX1435 results in alarge increase in C14:0 production (over 60% of the fatty acid profile).

Constructs used for the overexpression of the Cpal FATB2 gene were codonoptimized for expression in P. moriformis as described herein. Cupheapalustris FATB2 is a C14 preferring thioesterase. Two constructs, bothencoding the Cpal FATB2 gene, were prepared. The first construct,pSZ2479, can be written as6SA::CrTUB2-ScSUC2-CvNR:PmAMT3-CpSAD1tpExt-Cpa1FATB2ExtA-CvNR::6SB. TheFatB2 coding sequence is given as SEQ ID NO: 86 and the amino acidsequence is given as SEQ ID NO: 87. The second construct, pSZ2480 can bewritten as6SA::CrTUB2-ScSUC2-CvNR:PmAMT3-CpSAD1tpExt_Cpa1FATB2FLAG_ExtA-CvNR::6SB.The nucleic acid sequence and amino acid sequence are given as SEQ IDNO: 88 and SEQ ID NO: 89.

P. moriformis transformed with pSZ2480 produced high levels of myristicacid. The myristate content was 65.70 percent. This is a very largeincrease when compared to the myristate content of the wild-type oilproduced by the base strain, which has a myristate content ofapproximately 1%.

The fatty acid profile of the high myristate strain is shown in theTable 51 below.

TABLE 51 Fatty acid profile of high myristate strain. Fatty Acid % C10:00.04 C12:0 1.19 C14:0 65.7 C16:0 13.55 C18:0 0.57 C18:1 12.2 C18:2 5.13C20:0 0.05 C22:0 0.01 C24:0 0.01

Example 53 Production of Eicosenoic and Erucic Fatty Acids

In this example we demonstrate that expression of heterologous fattyacid elongase (FAE), also known as 3-ketoacyl-CoA synthase (KCS), genesfrom Cramble abyssinica (CaFAE, Accession No: AY793549), Lunaria annua(LaFAE, ACJ61777), and Cardamine graeca (CgFAE, ACJ61778) leads toproduction of very long chain monounsaturated fatty acids such aseicosenoic (20:1^(Δ11)) and erucic (22:1^(Δ13)) acids in classicallymutagenized derivative of UTEX 1435, Strain Z. On the other hand aputative FAE gene from Tropaeolum majus (TmFAE, ABD77097) and two FAEgenes from Brassica napus (BnFAE1, AAA96054 and BnFAE2, AAT65206), whileresulting in modest increase in eicosenoic (20:1^(Δ11)), produced nodetectable erucic acid in STRAIN Z. Interestingly the unsaturated fattyacid profile obtained with heterologous expression of BnFAE1 in STRAIN Zresulted in noticeable increase in Docosadienoic acid (22:2n6). All thegenes were codon optimized to reflect UTEX 1435 codon usage. Theseresults suggest that CaFAE, LaFAE or CgFAE genes encode condensingenzymes involved in the biosynthesis of very long-chain utilizingmonounsaturated and saturated acyl substrates, with specific capabilityfor improving the eicosenoic and erucic acid content.

Construct Used for the Expression of the Cramble Abyssinica Fatty AcidElongase (CaFAE) in P. moriformis (UTEX 1435 Strain STRAIN Z)—[pSZ3070]:

In this example STRAIN Z strains, transformed with the constructpSZ3070, were generated, which express sucrose invertase (allowing fortheir selection and growth on medium containing sucrose) and C.abyssinica FAE gene. Construct pSZ3070 introduced for expression inSTRAIN Z can be written as6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-CaFAE-Cvnr::6S.

The sequence of the transforming DNA is provided below. Relevantrestriction sites in the construct are indicated in lowercase, bold, andare from 5′-3′ BspQI, KpnI, XbaI, MfeI, BamHI, EcoRI, SpeI, AflII, SacI,BspQI, respectively. BspQI sites delimit the 5′ and 3′ ends of thetransforming DNA. Bold, lowercase sequences represent genomic DNA fromSTRAIN Z that permit targeted integration at the 6S locus via homologousrecombination. Proceeding in the 5′ to 3′ direction, the C. reinhardtiiβ-tubulin promoter driving the expression of the Saccharomycescerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, therebypermitting the strain to grow on sucrose) is indicated by lowercase,boxed text. The initiator ATG and terminator TGA for SUC2 are indicatedby uppercase italics, while the coding region is indicated withlowercase italics. The Chlorella vulgaris nitrate reductase (NR) gene 3′UTR is indicated by lowercase underlined text followed by an endogenousAMTS promoter of P. moriformis, indicated by boxed italicized text. TheInitiator ATG and terminator TGA codons of the CaFAE are indicated byuppercase, bold italics, while the remainder of the gene is indicated bybold italics. The C. vulgaris nitrate reductase 3′ UTR is againindicated by lowercase underlined text followed by the STRAIN Z 6Sgenomic region indicated by bold, lowercase text. The final constructwas sequenced to ensure correct reading frames and targeting sequences.

Nucleotide sequence of transforming DNA contained in plasmid pSZ3070:(SEQ ID NO: 102) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgt

atgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaaccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagttaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcac

actacgtgatcaccaacctgttcaacctgtgcttcttccccctgaccgccatcgtggccggcaaggcctcccgcctgaccatcgacgacctgcaccacctgtactactectacctgcagcacaacgtgatcaccatcgcccccctgttcgccttcaccgtgltcggctccatcctgtacatcgtgacccgccccaagcccgtgtacctggtggagtactcctgctacctgccccccacccagtgccgctcctccatctccaaggtgatggacatcttctaccaggtgcgcaaggccgaccecttccgcaacggcacctgcgacgactcctcctggctggacttcctgcgcaagatccaggagcgctccggcctgggcgacgagacccacggccccgagggcctgctgcaggtgcccccccgcaagaccttcgccgccgcccgcgaggagaccgagcaggtgatcgtgggcgccctgaagaacctgttcgagaacaccaaggtgaaccccaaggacatcggcatcctggtggtgaactcctccatgttcaaccccaccccctccctgtccgccatggtggtgaacaccttcaagctgcgctccaacgtgcgctccttcaacctgggcggcatgggctgctccgccggcgtgatcgccatcgacctggccaaggacctgctgcacgtgcacaagaacacctacgccctggtggtgtccaccgagaacatcacctacaacatctacgccggcgacaaccgctccatgatggtgtccaactgcctgttccgcgtgggcggcgccgccatcctgctgtccaacaagccccgcgaccgccgccgctccaagtacgagctggtgcacaccgtgcgcacccacaccggcgccgacgacaagtecttccgctgcgtgcagcagggcgacgacgagaacggcaagaccggcgtgtccctgtccaaggacatcaccgaggtggccggccgcaccgtgaagaagaacatcgccaccctgggccccctgatcctgcccctgtccgagaagctgctgttcttcgtgaccttcatggccaagaagctgttcaaggacaaggtgaagcactactacgtgcccgacttcaagctggccatcgaccacttctgcatccacgccggcggccgcgccgtgatcgacgtgctggagaagaacctgggcctggcccccatcgacgtggaggcctcccgctccaccctgcaccgcttcggcaacacctcctcctcctccatctggtacgagctggcctacatcgaggccaagggccgcatgaagaagggcaacaaggtgtggcagatcgccctgggctccggcttcaagtgcaactccgccgtgtgggtggccctgtccaacgtgaaggcctccaccaactccccctgggagcactgcatcgaccgctaccccgtgaagatcgactccgactccgccaagtccgagacccgcgcccagaacggccgctecTGActtaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctatatcaaacagcctcagtgtgtagatcttgtgtgtacgcgcattgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccaccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagac

Constructs Used for the Expression of the FAE Genes from Higher Plantsin STRAIN Z:

In addition to the CaFAE gene (pSZ3070), LaFAE (pSZ3071) from Lunariaannua, CgFAE (pSZ3072) from Cardamine graeca, TmFAE (pSZ3067) Tropaeolummajus and BnFAE1 (pSZ3068) and BnFAE2 (pSZ3069) genes from Brassicanapus have been constructed for expression in STRAIN Z. These constructscan be described as:

pSZ3071—6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-LaFAE-Cvnr::6SpSZ3072—6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-CgFAE-Cvnr::6SpSZ3067—6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-TmFAE-Cvnr::6SpSZ3068—6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-BnFAE1-Cvnr::6SpSZ3069—6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-BnFAE2-Cvnr::6S

All these constructs have the same vector backbone; selectable marker,promoters, and 3′ utr as pSZ3070, differing only in the respective FAEgenes. Relevant restriction sites in these constructs are also the sameas in pSZ3070. The sequences of LaFAE, CgFAE, TmFAE, BnFAE1 and BnFAE2are shown below. Relevant restriction sites as bold text including SpeIand AflII are shown 5′-3′ respectively.

Nucleotide sequence of LaFAE contained in pSZ3071: (SEQ ID NO: 103)

ATG

TGA

Nucleotide sequence of CgFAE contained in pSZ3072: (SEQ ID NO: 104)

ATG

TGA

Nucleotide sequence of TmFAE contained in pSZ3067: (SEQ ID NO: 105)

TGA

Nucleotide sequence of BnFAE1 contained in pSZ3068: (SEQ ID NO: 106)

ATG

A

Nucleotide sequence of BnFAE2 contained in pSZ3069: (SEQ ID NO: 107)

ATG

TGA

To determine their impact on fatty acid profiles, the above constructscontaining various heterologous FAE genes, driven by the PmAMT3promoter, were transformed independently into STRAIN Z.

Primary transformants were clonally purified and grown under standardlow-nitrogen lipid production conditions at pH 7.0 (all the plasmidsrequire growth at pH 7.0 to allow for maximal FAE gene expression whendriven by the pH regulated PmAMT03 promoter). The resulting profilesfrom a set of representative clones arising from transformations withpSZ3070, pSZ3071, pSZ3072, pSZ3067, pSZ3068 and pSZ3069 into STRAIN Zare shown in Tables 52-57, respectively, below.

All the transgenic STRAIN Z strains expressing heterologous FAE genesshow an increased accumulation of C20:1 and C22:1 fatty acid (see Tables52-57). The increase in eicosenoic (20:1^(Δ11)) and erucic (22:1^(Δ13))acids levels over the wildtype is consistently higher than the wildtypelevels. Additionally, the unsaturated fatty acid profile obtained withheterologous expression of BnFAE1 in STRAIN Z resulted in noticeableincrease in Docosadienoic acid (C22:2n6). Protein alignment ofaforementioned FAE expressed in STRAIN Z is shown in FIG. 23.

TABLE 52 Unsaturated fatty acid profile in STRAIN Z and representativederivative transgenic lines transformed with pSZ3070 (CaFAE) DNA. SampleID C18:1 C18:2 C18:3a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 51.499.13 0.65 4.35 1.24 0.11 0.00 D1828-20 STRAIN Z; T588; 55.59 7.65 0.503.78 0.85 0.00 0.13 D1828-23 STRAIN Z; T588; 54.70 7.64 0.50 3.44 0.850.09 0.00 D1828-43 STRAIN Z; T588; 52.43 7.89 0.59 2.72 0.73 0.00 0.00D1828-12 STRAIN Z; T588; 56.02 7.12 0.52 3.04 0.63 0.10 0.11 D1828-19Cntrl STRAIN Z 57.99 6.62 0.56 0.19 0.00 0.06 0.05 pH 7 Cntrl STRAIN Z57.70 7.08 0.54 0.11 0.00 0.05 0.05 pH 5

TABLE 53 Unsaturated fatty acid profile in STRAIN Z and representativederivative transgenic lines transformed with pSZ3071 (LaFAE) DNA. SampleID C18:1 C18:2 C18:3a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 54.667.04 0.52 1.82 0.84 0.12 0.09 D1829-36 STRAIN Z; T588; 56.27 6.72 0.511.70 0.72 0.09 0.00 D1829-24 STRAIN Z; T588; 56.65 8.36 0.54 2.04 0.670.00 0.00 D1829-11 STRAIN Z; T588; 55.57 7.71 0.53 0.10 0.66 0.00 0.00D1829-35 STRAIN Z; T588; 56.03 7.06 0.54 1.54 0.51 0.06 0.08 D1829-42Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.00 0.06 0.05 pH 7 Cntrl STRAIN Z57.99 6.62 0.56 0.19 0.00 0.05 0.05 pH 5

TABLE 54 Unsaturated fatty acid profile in STRAIN Z and representativederivative transgenic lines transformed with pSZ3072 (CgFAE) DNA. SampleID C18:1 C18:2 C18:3a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 57.747.79 0.52 1.61 0.25 0.11 0.05 D1830-47 STRAIN Z; T588; 58.06 7.39 0.551.64 0.22 0.07 0.06 D1830-16 STRAIN Z; T588; 57.77 6.86 0.51 1.34 0.190.09 0.00 D1830-12 STRAIN Z; T588; 58.45 7.54 0.49 1.65 0.19 0.06 0.00D1830-37 STRAIN Z; T588; 57.10 7.28 0.56 1.43 0.19 0.07 0.00 D1830-44Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.00 0.06 0.05 pH 7 Cntrl STRAIN Z57.99 6.62 0.56 0.19 0.00 0.05 0.05 pH 5

TABLE 55 Unsaturated fatty acid profile in Strain AR and representativederivative transgenic lines transformed with pSZ3070 (TmFAE) DNA. Nodetectable Erucic (22:1) acid peaks were reported for these transgeniclines. Sample ID C18:1 C18:2 C18:3a C20:1 C22:2n6 C22:5 STRAIN Z; T588;59.97 7.44 0.56 0.57 0.00 0.00 D1825-47 STRAIN Z; T588; 58.77 7.16 0.510.50 0.09 0.11 D1825-35 STRAIN Z; T588; 60.40 7.82 0.47 0.44 0.07 0.07D1825-27 STRAIN Z; T588; 58.07 7.32 0.54 0.41 0.05 0.05 D1825-14 STRAINZ; T588; 58.66 7.74 0.46 0.39 0.08 0.00 D1825-40 Cntrl STRAIN Z 57.996.62 0.56 0.19 0.05 0.05 pH 7 Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.060.05 pH 5

TABLE 56 Unsaturated fatty acid profile in STRAIN Z and representativederivative transgenic lines transformed with pSZ3068 (BnFAE1) DNA. Nodetectable Erucic (22:1) acid peaks were reported for these transgeniclines. Sample ID C18:1 C18:2 C18:3a C20:1 C22:2n6 C22:5 STRAIN Z; T588;59.82 7.88 0.55 0.32 0.17 0.10 D1826-30 STRAIN Z; T588; 59.32 8.02 0.580.27 0.18 0.07 D1826-23 STRAIN Z; T588; 59.63 7.49 0.55 0.27 0.19 0.08D1826-45 STRAIN Z; T588; 59.35 7.78 0.57 0.26 0.23 0.00 D1826-24 STRAINZ; T588; 59.14 7.61 0.57 0.25 0.22 0.05 D1826-34 Cntrl STRAIN Z 57.817.15 0.59 0.19 0.04 0.06 pH 7 Cntrl STRAIN Z 58.23 6.70 0.58 0.18 0.050.06 pH 5

TABLE 57 Unsaturated fatty acid profile in STRAIN Z and representativederivative transgenic lines transformed with pSZ3069 (BnFAE2) DNA. Nodetectable Erucic (22:1) acid peaks were reported for these transgeniclines. Sample ID C18:1 C18:2 C18:3a C20:1 C22:2n6 C22:5 STRAIN Z; T588;60.59 8.20 0.57 0.34 0.00 0.07 D1827-6 STRAIN Z; T588; 59.62 6.44 0.520.30 0.07 0.00 D1827-42 STRAIN Z; T588; 59.71 7.99 0.59 0.30 0.06 0.00D1827-48 STRAIN Z; T588; 60.66 8.21 0.59 0.29 0.04 0.00 D1827-43 STRAINZ; T588; 60.26 7.99 0.57 0.28 0.04 0.00 D1827-3 Cntrl STRAIN Z 57.817.15 0.59 0.19 0.04 0.06 pH 7 Cntrl STRAIN Z 58.23 6.70 0.58 0.18 0.050.06 pH 5

Example 54 Elevating Total Unsaturated FATY Acids Level by ExpressingHeterologous Desaturase Genes

One of the approaches to generate a “zero SAT FAT” (e.g., totalunsaturated fatty acids target at 97% or more/less than or equal to 3%saturated fat) strain is to express desaturase genes in a high oleicstrain such as Strain N, which we found to produce about 85% C18:1 withtotal un-saturates around 93% in multiple fermentation runs. Weinvestigated if the total saturates will be further diminished byexpressing desaturase genes in Strain N.

In the examples below, we demonstrated the ability to reduce stearic andpalmitic levels in wild type strain UTEX1435 by over expression ofheterologous stearoyl-ACP desaturase genes, including desaturases fromOlea europaea, Ricinus communis, and Chlorella protothecoides.

Construct used for the expression of the Olea europaea stearoyl-ACPdesaturase: To introduce the O. europaea stearoyl-ACP desaturase(Accession No: AAB67840.1) into UTEX1435, Strain A, the Saccharomycescerevisiae invertase gene was utilized as the selectable marker toconfer the ability of growing on sucrose media. The construct that hasbeen expressed in UTEX1435, Strain A can be written as6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:OeSAD:CvNR::65B and is termedpSZ1377.

Relevant restriction sites in the construct pSZ1377 are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene is indicated by boxedtext. The initiator ATG and terminator TGA for invertase are indicatedby uppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by the second C.reinhardtii β-tubulin promoter driving the expression of the OeSAD,indicated by boxed italics text. The Initiator ATG and terminator TGAcodons of the OeSAD are indicated by uppercase, bold italics, while theremainder of the stearoyl-ACP desaturase coding region is indicated bybold italics. The Chlorella protothecoides stearoyl-ACP desaturasetransit peptide is located between initiator ATG and the Asc I site. TheC. vulgaris nitrate reductase 3′ UTR is again indicated by lowercaseunderlined text followed by the 6S genomic region indicated by bold,lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ 1377:(SEQ ID NO: 108) gctcttcgccgccgccactectgetcgagegcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctegtgcgcgtcgctgatgtecatcaccaggtecatgaggtagccttgegccggctgagccactgettcgtecgggeggccaagaggagcatgagggaggactectggtccagggtcctgacgtggtcgcggetctgggagegggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtegccgccgaccaggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtetaggcagaatecctaccagtcatggctttacctggatgacggcctgcgaacagctgtecagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagegccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcc

cgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtagggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccaguccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagaggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccagcagaccttatcaacaccgacccgacctacgggagcgccagggcatcgcgtgggcctccaactgggagtactccgcatcgtgcccaccaacccaggcgctectccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccaggagttcgagaggtgtacgccgtcaacaccacccagacgataccaagtccgtgttcgcggacctctccactggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtectcatatcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagccatcaagagcgagaacgacctgtcctactacaaggtgtacggettgctggaccagaacatcctggagagtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccagggctccgtgaacatgacgacgggggtggacaacctgactacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcattatcaaacagcctcagtgtgatgatcagtgtgtacgcgcttttgcgagagctagctgcagtgctatagcgaataccacccccagcatccccaccctcgatcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccaggtagggctccgcctgtaactcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggacacacacgtgccacgaggcgaggtggcaggtgacaatgatcggtggagctgatg

ggcgcccagcgaggcccctccccgtgcgcgggcgcgagaggtgcacgtgcaggtgacccactccctggcccccgagaagcgcgagatcttcaactccctgaacaactgggcccaggagaacatcctggtgctgctgaaggacgtggacaagtgctggcagccctccgacttcctgcccgactccgcctccgagggcttcgacgagcaggtgatggagctgcgcaagcgctgcaaggagatccccgacgactacttcatcgtgctggtgggcgacatgatcaccgaggaggccctgcccacctaccagaccatgctgaacaccctggacggcgtgcgcgacgagaccggcgcctccctgaccccctgggccatctggacccgcgcctggaccgccgaggagaaccgccacggcgacctgctgaacaagtacctgtacctgtccggccgcgtggacatgaagcagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccgagaacaaccectacctgggcttcatctacacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgcctggccaaggagcacggcgacctgaagctggcccagatctgcggcatcatcgccgccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccccgacggcaccgtgctggccctggccgacatgatgcgcaagaaggtgtccatgcccgcccacctgatgtacgacggccaggacgacaacctgttcgagaacttctectccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagttcctggtgggccgctgggacatcgagaagctgaccggcctgtccggcgagggccgcaaggcccaggactacgtgtgcaccctgcccccccgcatccgccgcctggaggagcgcgcccagtcccgcgtgaagaaggcctccgccacccccttctcctggatcttcggccgcgagatcaacctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGA atcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgagccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggatgggaacacaaatggaaagccttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagettggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgeggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

Construct Used for the Expression of the Ricinus communis Stearoyl-ACPDesaturase:

To introduce the Ricinus communis stearoyl-ACP desaturase (Accession No:AAA74692.1) into UTEX1435, Strain A, the Saccharomyces cerevisiaeinvertase gene was utilized as the selectable marker to confer theability of growing on sucrose media. The construct that has beenexpressed in UTEX1435, Strain A can be written as6SA::CrTUB2:ScSUC2:CvNR::PmAMT03:CpSADtp:RcSAD:CvNR::6SB and is termedpSZ1454.

Relevant restriction sites in the construct pSZ1454 are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA that permit targetedintegration at 6s nuclear chromosomal locus via homologousrecombination. Proceeding in the 5′ to 3′ direction, the C. reinhardtiiβ-tubulin promoter driving the expression of the yeast sucrose invertasegene is indicated by boxed text. The initiator ATG and terminator TGAfor invertase are indicated by uppercase, bold italics while the codingregion is indicated in lowercase italics. The Chlorella vulgaris nitratereductase 3′ UTR is indicated by lowercase underlined text followed bythe endogenous AMT03 promoter driving the expression of the RcSAD,indicated by boxed italics text. The Initiator ATG and terminator TGAcodons of the RcSAD are indicated by uppercase, bold italics, while theremainder of the stearoyl-ACP desaturase coding region is indicated bybold italics. The Chlorella protothecoides stearoyl-ACP desaturasetransit peptide is located between initiator ATG and the Asc I site. TheC. vulgaris nitrate reductase 3′ UTR is again indicated by lowercaseunderlined text followed by the 6S genomic region indicated by bold,lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ1454:(SEQ ID NO: 109) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgccc

gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagaccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggt

ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcg ggcgcgccgcctccaccctgaagtccggctccaaggaggtggagaacctgaagaagcccttcatgcccccccgcgaggtgcacgtgcaggtgacccactccatgcccccccagaagatcgagatcttcaagtccctggacaactgggccgaggagaacatcctggtgcacctgaagcccgtggagaagtgctggcagccccaggacttcctgcccgaccccgcctccgacggcttcgacgagcaggtgcgcgagctgcgcgagcgcgccaaggagatccccgacgactacttcgtggtgctggtgggcgacatgatcaccgaggaggccctgcccacctaccagaccatgctgaacaccctggacggcgtgcgcgacgagaccggcgcctcccccacctcctgggccatctggacccgcgcctggaccgccgaggagaaccgccacggcgacctgctgaacaagtacctgtacctgtccggccgcgtggacatgcgccagatcgagaagaccatccagtacctgatcggctccggcatggacccccgcaccgagaactccccctacctgggcttcatctacacctccttccaggagcgcgccaccttcatctcccacggcaacaccgcccgccaggccaaggagcacggcgacatcaagctggcccagatctgcggcaccatcgccgccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccccgacggcaccgtgctggccttcgccgacatgatgcgcaagaagatctccatgcccgcccacctgatgtacgacggccgcgacgacaacctgttcgaccacttctccgccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagttcctggtgggccgctggaaggtggacaagctgaccggcctgtccgccgagggccagaaggcccaggactacgtgtgccgcctgcccccccgcatccgccgcctggaggagcgcgcccagggccgcgccaaggaggcccccaccatgcccttctcctggatcttcgaccgccaggtgaagctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGAat cgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctc ttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

Construct Used for the Expression of the Chlorella protothecoidesStearoyl-ACP Desaturase:

To introduce the Chlorella protothecoides stearoyl-ACP desaturase intoUTEX1435, Strain Z, the Saccharomyces cerevisiae invertase gene wasutilized as the selectable marker to confer the ability of growing onsucrose media. The construct that has been expressed in UTEX1435, StrainZ can be written as 6SA::CrTUB2:ScSUC2:CvNR::PmAMT03:CpSAD1:CvNR::6SBand is termed pSZ3144.

Relevant restriction sites in the construct pSZ3144 are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQIsites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene is indicated by boxedtext. The initiator ATG and terminator TGA for invertase are indicatedby uppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by the endogenous AMT03promoter driving the expression of the CpSAD1, indicated by boxeditalics text. The Initiator ATG and terminator TGA codons of the CpSAD1are indicated by uppercase, bold italics, while the remainder of thestearoyl-ACP desaturase coding region is indicated by bold italics. TheC. vulgaris nitrate reductase 3′ UTR is again indicated by lowercaseunderlined text followed by the 6S genomic region indicated by bold,lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3144:(SEQ ID NO: 110) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccaggccattcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtagccagegccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagegggccagcatcatctggctctgccgcaccgaggccgcctccaactggtectccagcagccgcagtegccgccgaccaggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccagtetaggcagaatecctaccagtcatggctttacctggatgacggcctgcgaacagetgtccagcgaccctcgctgccgccgcactcccgcacgcactaccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccagcgcgttagtgagccatccatgcagaccggtgagagccgacttgagtgcgccaccccccacaccacctcctcccagaccaattctgtcaccataggegaaggcatcggcctcggcc

cgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtagggggacgcccttgactggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcgccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtectggaagaggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagteccgcgtggtggacttcggcaaggactactacgccagcagaccttatcaacaccgacccgacctacgggagcgccagggcatcgcgtgggcctccaactgggagtactccgcatcgtgcccaccaacccaggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccaggagttcgagaggtgtacgccgtcaacaccacccagacgataccaagtccgtgttcgcggacctctccactggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtectcatatcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagccatcaagagcgagaacgacctgtcctactacaaggtgtacggettgctggaccagaacatcctggagagtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccagggctccgtgaacatgacgacgggggtggacaacctgactacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgagccgccacacttgctgccttgacctgtgaatatccctgccgcattatcaaacagcctcagtgtgatgatcagtgtgtacgcgcttttgcgagagctagctgatgtgctatttgcgaataccacccccagcatcccatccctcgatcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccaggtagggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggacttcgtccattagcgaagcgtccggacacacacgtgccacgaggcgaggtggcaggtgacaatgatcggtggagctgatg

ctccaccttctccgccttcaacgcccgctgcggcgacctgcgccgctccgccggctccggcccccgccgccccgcccgccccctgcccgtgcgcgccgccatcgcctccgaggtgcccgtggccaccacctccccccgccccggccccaccgtgtactccaagctggacaaggcccacaccctgacccccgagcgcatggagctgatcaacggcatgtccgccttcgccgaggagcgcatcctgcccgtgctgcagcccgtggagaagctgtggcagccccaggacctgctgcccgaccccgagtcccccgacttcctggaccaggtggccgagctgcgcgcccgcgccgccaacgtgcccgacgactacttcgtggtgctggtgggcgacatgatcaccgaggaggccctgcccacctacatggccatgctgaacaccctggacggcgtgcgcgacgagaccggcgccgccgaccacccctggggccgctggacccgccagtgggtggccgaggagaaccgccacggcgacctgctgaacaagtactgctggctgaccggccgcgtgaacatgaaggccatcgaggtgaccatccagaacctgatcggctccggcatgaaccccaagaccgagaacaacccctacctgggcttcgtgtacacctccttccaggagcgcgccaccaagtactcccacggcaacaccgcccgcctggccgcccagtacggcgacgccaccctgtccaaggtgtgcggcgtgatcgccgccgacgagggccgccacgagatcgcctacacccgcatcgtggaggagttatccgcctggaccccgagggcgccatgtccgcctacgccgacatgatgcgcaagcagatcaccatgcccgcccacctgatggacgaccagcagcacggcacccgcaacaccggccgcaacctgttcgccgacttctccgccgtgaccgagaagctggacgtgtacgacgccgaggactactgcaagatcctggagcacctgaactcccgctggaagatcgccgaccgcaccgtgtccggcgacgccggcgccgaccaggagtacgtgctgcgcctgccctcccgcttccgcaagctggccgagaagtccgccgccaagcgcgccaagaccaagcccaagcccgtggccttctcctggctgtccggccgcgaggtgatggtgTGA atcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgattcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttg aagagc

Primary transformants were clonally purified and grown underlow-nitrogen lipid production conditions at either pH 5.0 or pH 7.0,depending on the promoters that drive the expression of the desaturasegenes. Transgenic lines arising from the transformations with pSZ1377(D583) were assayed in (low-nitrogen) lipid production media at pH 5.0,because of the nature of the promoters and the fact that P. moriformisproduces more lipid at pH 5.0. Transgenic lines generated from thetransformation of pSZ1454 (D648) and pSZ3144 (D1923) are assayed at pH7.0 to allow for maximal desaturase gene expression when driven by thepH regulated PmAMT3 promoter. The resulting profiles from representativeclones arising from transformations with D583, D648, and D1923 are shownin Tables 58, 59, and 60, respectively, below. The result of expressionof OeSAD and CpSAD1 genes is a clear diminution of C18:0 chain lengthswith an increase in C18:1. Also we noticed that there is a subtleincrease in the level of C16:1, indicating these stearoyl-ACPdesaturases may have broad specificity. The transformants resulted fromthe expression of RcSAD gene also diminishes in the level of C18:0, andelevation in C16:1. Notably, C16:1 could be increased from under 1% toover 1.5% or over 2%. However, there is also a drop in the level ofC18:1 fatty acid and increase in C18:2, which may be caused by thegrowth defect of these transgenic lines.

TABLE 58 Lipid profile of representative clones arising fromtransformation with D583 (pSZ1377) DNA Sample ID C16:0 C16:1 C18:0 C18:1C18:2 D583-25 19.20 1.53 1.15 64.08 11.76 D583-10 21.86 0.99 1.77 61.4311.42 D583-3 21.94 0.95 1.85 62.22 10.53 D583-33 20.76 0.95 1.85 61.7612.17 D583-26 20.18 0.92 1.89 62.56 11.97 D583-1 21.28 0.95 1.90 62.6310.94 S1331 25.48 0.71 3.23 59.70 8.25

TABLE 59 Lipid profile of representative clones arising fromtransformation with D648 (pSZ1454) DNA Sample ID C16:0 C16:1 C18:0 C18:1C18:2 D648-9 26.92 2.30 1.12 54.27 11.30 D648-28 26.54 2.50 1.32 52.5812.90 D648-15 29.47 1.68 1.48 51.74 11.48 D648-12 27.39 1.41 1.66 54.4511.58 D648-43 29.74 1.52 1.68 52.59 10.85 D648-7 26.98 1.62 1.69 54.5111.39 S1331-pH 7 25.86 0.96 2.84 58.33 9.16

TABLE 60 Lipid profile of representative clones arising fromtransformation with D1923 (pSZ3144) DNA Sample ID C14:0 C14.1 C16:0C16:1 C18:0 C18:1 C18:2 Block 2; E2; pH7; 1.46 0.11 20.74 2.54 0.8663.99 9.03 STRAIN Z; T613; D1923-2 Block 2; G12; pH7; 1.52 0.10 25.201.97 1.67 61.10 7.38 STRAIN Z; T613; D1923-36 Block 2; E8; pH7; 1.480.09 26.41 1.78 1.54 60.54 7.01 STRAIN Z; T613; D1923-8 Block 2; F3;1.50 0.07 25.87 1.75 1.62 61.25 6.94 pH7STRAIN Z; T613; D1923-15 Block2; F9; pH7; 1.47 0.07 27.02 1.73 1.84 60.15 6.55 STRAIN Z; T613;D1923-21 Block 2; F4; pH7; 1.44 0.07 24.30 1.71 1.41 62.79 7.29 STRAINZ; T613; D1923-16 pH7 STRAIN Z 1.47 0.00 28.25 0.82 3.16 58.27 6.72

Example 55 Microemulsion Preparation and Cleaning Efficiency

Microemulsions were prepared using the fatty acid methyl ester of thehigh oleic (HO) oil of Example 49 (“FAME-HO oil” below), d-Limonene, andvarious other methyl esters, and the performance/ability of themicroemulsions to remove bitumen soil from a hard surface were compared.Eight microemulsions were tested, with varying percentages ofcomponents, as shown in Table 61, below.

TABLE 61 Microemulsion compositions. 1 2 3 4 5 6 7 8 d-Limonene 20.0 x xx x x x 10.0  FAME-HO x 20.0  10.0 15.0  x x 10.0  10.0  oil Methyl x xx x 20.0  x 10.0  x Soyate Methyl x x x x x 20.0  x x Canolate Methyl xx x 5.0 x x x x Ricinoleate Videt 13.5 20.72 15.9 19.24 20.72 20.7220.72 19.24 ME-80 Neodol  3.0 3.0  3.0 3.0 3.0 3.0 3.0 3.0 N91-8 diWater 63.5 56.28 71.1 57.76 56.28 56.28 56.28 57.76

Each of the compositions in Table 61 was tested for cleaning efficacy.Reflectance values of white vinyl tile were taken (via 5 readings pertile in the 3×2 inch test area) before soiling, and an averagereflectance calculated. A bitumen/solvent soil mixture was applied tothe white vinyl tile and rubbed to an even consistency. The soil wasthen dried for 17.5 hours in a 40° C. forced air oven. Once cooled toambient temperature, the reflectance values were again measured and thesoiled panel was placed on a Gardner straight-line washabilityapparatus. A 10 ml sample of test product (microemulsion compositions)was applied to a cellulose sponge and inverted directly onto the soiledtest area. The panel was scrubbed for 3 cycles and then rinsed with cooltap water and air dried. Reflectance after cleaning was determined andused to calculate percent soil removed. The cleaning efficiency resultsare shown in Table 62, below. % cleaningefficiency=(R^(c)−R^(s))/(R^(o)−R_(s))×100, where R^(c) is the cleanedreflectance, R^(o) is the original reflectance, and R^(s) is the soiledreflectance.

TABLE 62 Cleaning efficiency results of microemulsion compositions.Microemulsion Composition Average % Cleaning (from Table 61) Efficiency1 73.8 2 77.7 3 11.2 4 51.0 5 75.1 6 76.3 7 76.7 8 78.4

Example 56 Epoxidized Oil from Strain R

To a 2 L 3-neck round bottom flask equipped with a mechanical stirrer,addition funnel, heating mantle, thermocouple and Argon gas inlet wascharged 787.95 g of oil from Strain R (Example 49) and 32 mL of formicacid. The reaction mixture was heated to 50° C. and 179 mL of 50%hydrogen peroxide solution was added dropwise over 12 minutes whilemaintaining the temperature below 65° C. An ice water bath was used inplace of the heating mantle to maintain the temperature between 50° C.and 65° C. After observing an exotherm, the reaction was then maintainedat 55° C. with periodic sampling and determination by proton NMR thatthe reaction was >95% complete. The reaction mixture was transferred toa 6 L separatory funnel with 2 L of ethyl acetate, water layer removed,treated with 6×150 mL of saturated sodium bicarbonate at which point thepH of the water layer was 8-9 and followed with a brine rinse. Theepoxidized oil was dried over magnesium sulfate and filtered warm due tolow melting solid product. The solvent was removed at 60° C. underreduced pressure to yield 814 g (98.5% yield) of epoxidized oil.

Example 57 Epoxide Ring Opening

To a 3 L 3-neck round bottom flask equipped with a mechanical stirrer,thermocouple, addition funnel, heating mantle and Argon gas inlet wascharged 500 mL of methanol and 21.20 g of 48% fluoroboric acid. Themixture was stirred and heated to 40° C. Over a 1.5 hour time period, amixture of 915.60 g of epoxidized algal oil from Example 56 and 400 mLof chloroform was added dropwise to the reaction flask. An additional120 mL of chloroform was used to rinse the remaining oil into thereaction flask. After 1 hour, the reaction mixture was sampled andoxirane conversion determined to be complete by proton NMR. To thereaction flask, 11 g of sodium bicarbonate was added with stirring toneutralize the acid. A majority of the solvent was removed under reducedpressure at 40° C. The polyol was then dissolved in 1 L of ethyl acetateand extracted with 3×100 mL of a 20% potassium carbonate solution. Theorganic layer was then dried over magnesium sulfate, filtered andsolvent removed at 80° C. under reduced pressure to yield 951.71 g(95.7% yield) of the methyl ether alcohol. The hydroxyl value wasdetermined by NMR to be 146 mg KOH/g. The product was then combined witha similarly prepared batch (1955 g, 93.9% yield, HV_(NMR)=145) toconstitute the final methyl ether alcohol (HV_(NMR)=145 andHV_(Titration)=145). Alternatively, the fluoroboric acid can be replacedwith copper tetrafluoroborate. This replacement was found to result in anumber of advantages including allowing for the addition of a fullcharge of reagents instead of dropwise addition, less diglycerideformation, eliminating need for acid neutralization and resulting saltremoval, extraction of catalyst with water/brine wash, and diminishedreaction exotherm allowing for less solvent use. The ethyl acetate inthe workup can also be replaced with methyl acetate. This replacementwas found to result in less emulsification and easier separation.Additionally removal of the solvent was not necessary and can be carriedon to the urethane condensation reaction.

Example 58 Epoxidized Oil from High Oleic Strain

A classically mutagenized P. moriformis line was transformed with aconstruct targeting an endogenous FATA1 site for disruption withoverexpression of an endogenous KASII gene using the methods describedabove. The resulting strain produced an oil with a high oleic acidprofile as shown in Table 63 below.

TABLE 63 Fatty acid profile of oil. Fatty acid profile Percent C10:00.01 C12:0 0.03 C14:0 0.45 C15:0 0.02 C16:0 3.34 C16:1 cis-Δ7 0.05 C16:1cis-Δ9 0.11 C17:0 0.04 C17:1 cis-Δ9 0.03 C18:0 2.82 C18:1 81.72 C18:2cis, cis-Δ9, Δ12 8.3 C18:2 trans, trans-Δ9, Δ12 0.04 C18:3 alpha 0.48C20:0 0.33 C20:1 0.87 C22:0 0.08 C23:0 0.02 C24:0 0.17

To a 5 L 3-neck round bottom flask equipped with a mechanical stirrer,addition funnel, heating mantle was charged 2595 g of oil from the higholeic strain and 123 mL of formic acid, thermocouple and Argon gasinlet. The reaction mixture was heated to 50° C. and 681 mL of 50%hydrogen peroxide solution was added dropwise over 1 hour whilemaintaining the temperature below 55° C. An ice water bath was used inplace of the heating mantle to maintain the temperature between 50° C.and 65° C. After observing an exotherm, the reaction was then maintainedat 55° C. with periodic sampling and determination by proton NMR thatthe reaction was >95% complete. The reaction mixture was transferred toa 6 L separatory funnel with 2 L of ethyl acetate, water layer removedand then treated with 6×150 mL of saturated sodium bicarbonate at whichpoint the pH of the water layer was 8-9. The epoxidized oil solution wasdried over magnesium sulfate and filtered warm due to low melting solidproduct. The product was combined with another similarly prepared batchand solvent was removed at 60° C. under reduced pressure to yield 5295 g(98.2% yield) of epoxidized algal oil.

Example 59 Epoxide Ring Opening

To a 5 L 3-neck round bottom flask equipped with a mechanical stirrer,thermocouple, addition funnel, heating mantle and Argon gas inlet wascharged 1053 mL of methanol and 45.79 g of 48% fluoroboric acid. Themixture was stirred and heated to 40° C. Over a 2 hour time period, amixture of 2000 g of epoxidized algal oil from Example 58 and 1350 mL ofchloroform was added dropwise to the reaction flask. After 2.75 hours,the heating was stopped and the reaction mixture was sampled and oxiraneconversion determined to be complete by proton NMR. To the reactionflask, 25 g of sodium bicarbonate was added with stirring to neutralizethe acid. A majority of the solvent was removed under reduced pressureat 40° C. The alcohol was dissolved in 2 L of ethyl acetate, extractedwith 5×200 mL of a 20% potassium carbonate solution and followed by abrine wash. The organic layer was then dried over magnesium sulfate,filtered and solvent removed at 60° C. under reduced pressure to yield2159.37 g (89.4% yield) of alcohol. The hydroxyl value was determined byNMR to be 179 mg KOH/g. The product was combined with a similarlyprepared batch (2130.9 g, 88.2% yield, HV_(NMR)=179) to constitute thefinal batch (HV_(NMR)=179 and HV_(Titration)=184).

Example 60 Epoxidized Soybean Oil

To a 5 L 3-neck round bottom flask equipped with a mechanical stirrer,addition funnel, heating mantle, thermocouple and Argon gas inlet wascharged 2273 g of soybean oil (Sigma Aldrich Cat #W530261-5KG-K, Lot#MKAA3696V) and 150 mL of formic acid. The reaction mixture was heatedto 50° C. and 845 mL of 50% hydrogen peroxide solution was addeddropwise over 1.75 hours while maintaining the temperature at ˜55° C. Anice water bath was used in place of the heating mantle to maintain thetemperature between 50° C. and 65° C. After observing an exotherm, thereaction was then maintained at 55° C. with periodic sampling anddetermination by proton NMR that the reaction was >95% complete. Thereaction mixture was transferred to a 6 L separatory funnel with 2 L ofethyl acetate. The water layer was removed and polyol solution treatedwith 6×150 mL of saturated sodium bicarbonate at which point the pH ofthe aqueous layer was 8-9. The epoxidized oil was dried over magnesiumsulfate and filtered warm due to low melting solid product. The productsolution was combined with another similarly prepared batch, solventremoved at 60° C. under reduced pressure to yield 5231 g (101% yield,residual solvent noted in proton NMR spectrum) of epoxidized soybeanoil.

Example 61 Epoxidized Soybean Oil Ring Opening

To a 5 L 3-neck round bottom flask equipped with a mechanical stirrer,thermocouple, addition funnel, heating mantle and Argon gas inlet wascharged 1310 mL of methanol and 48.49 g of 48% fluoroboric acid. Themixture was stirred and heated to 40° C. Over a 2 hour time period, amixture of 1805 g of epoxidized soybean oil from Example 60 and 1250 mLof chloroform was added dropwise to the reaction flask. An additional310 mL of chloroform was used to rinse the remaining oil into thereaction flask. After 1 hour, the reaction mixture was sampled and theoxirane conversion was determined to be complete by proton NMR. 25 g ofsodium bicarbonate was added with stirring to neutralize the acid. Amajority of the solvent was removed under reduced pressure at 40° C. Thepolyol was dissolved in 2 L of ethyl acetate and extracted with 3×200 mLof a 20% potassium carbonate solution. The organic layer was then driedover magnesium sulfate, filtered and solvent removed at 90° C. underreduced pressure to yield 1839 g (89.5% yield) of polyol. The hydroxylvalue was determined by NMR to be 191 mg KOH/g. The product was combinedwith another similarly prepared batch (1909 g, 92.8% yield,HV_(NMR)=191) to constitute the final product (HV_(NMR)=191 andHV_(Titration)=201).

Example 62 Triglyceride/Polyol Analysis

The iodine values the triacylglyceride oils of Examples 56, 58, and 60and hydroxyl values of their corresponding polyols of Examples 57, 59,and 61 are shown below in Table 64.

TABLE 64 Iodine values of triglyceride oils. Triacylglyceride IodinePolyol Hydroxyl value Value (mg KOH/g) Sample NMR Titration NMRTitration Examples 56/57 74.3 74.6 145 145 (HSAO High Stability AlgalOil Examples 58/59 87.1 88 179 184 (HOAO High Oleic Algal Oil) Example60/61 122 — 191 201 (Soybean Oil)

The polyol of Example 57 was surprisingly found to be a liquid at roomtemperature and amenable to being a reactant with an isocyanate. US2011/006581 describes partially hydrogenated soybean oils. The oils withlow iodine values of 60 and 75 (Table 1, PHSBO-60 and PHSBO-75) were notable to be made into a foam because of the large amounts of solidspresent in the polyol (page 10, column 2 lines 18-20). Without beingbound by theory, it is believed that the partial hydrogenation processcreates trans-fatty acids and increases C18:0 levels throughhydrogenation of the C18:1, C18:2, and C18:3 double bonds, therebycreating triacylglycerides that favor solid formation particularly atelevated hydrogenation levels such as those where the iodine values ofthe triacylglyceride precursor is 75 or less.

Example 63 General Procedure for Preparation of Polyurethanes

Hand mixing was performed with a Craftsman ⅔ hp drill press with astainless steel stirring shaft and propeller blade operating at 3100 rpm

-   -   1. Weigh formulation components into plastic cups:        -   a. “A” Side—isocyanate        -   b. “B” Side—alcohol, catalysts, blowing agent, surfactant            and other additives (e.g., flame retardant)    -   2. “B” side is blended for 30 seconds    -   3. “A” side is added to the “B” cup and blended for a        predetermined amount of time (e.g., 10 seconds) and then poured        into the receiving container (e.g., plastic bin or paper cake        box)    -   4. Record reaction profile times in seconds: mix, cream, gel and        rise        -   a. Cream—mixture turns “cloudy”        -   b. Gel—“stringing” of the surface after being touched        -   c. Rise—foam has reached maximum height    -   5. Immediately place in 80° C. oven for 30 minutes to complete        cure    -   6. Remove from oven, crush foam by hand, inspect for deformity        and condition at standard room conditions for specified period        (e.g., 1 week).    -   7. Cut foam bun into required specimen shapes with a scalloped        blade on a band saw, removing edges and noting the rise        direction

Example 64 Polyurethane Foams

Polyurethane foams were prepared using polyols of Examples 57, 59, and61 according to the general procedure in Example 62 and the formulationsof Tables 65 and 66. The polyether polyol Jeffol G31-28 is a glycerininitiated, EO-tipped polyol, MW 6000.

Foams where 50% of the polyether polyol was replaced with algal derivedpolyol were found to have increased stability when the amount ofdiethanolamine catalyst was increased.

TABLE 65 Foam formulations. Eq. Polyol weight Control 1 2 3 4 5 JeffolG31-28 1951.54 97 87.3 87.3 87.3 77.6 77.6 Ex. 57 HSAO 386.9 0 9.7 0 019.4 0 Ex. 59 HOAO 313.4 0 0 9.7 0 0 19.4 Ex. 61 Soybean 293.7 0 0 0 9.70 0 % Replacement 0 10 10 10 20 20 Water 9 3.6 3.6 3.6 3.6 3.6 3.6Lumulse POE 26 416.2 3 3 3 3 3 3 Tegostab B 1335.7 1 1 1 1 1 1 4690Dabco 33LV 105 0.8 0.8 0.8 0.8 0.8 0.8 Diethanolamine 35.04 1 1 1 1 1 1Niax A-1 233.7 0.1 0.1 0.1 0.1 0.1 0.1 Isocyanate 130 57.81 60.15 60.8461.08 62.51 63.89 Mondur 1488 NCO Index 90 90 90 90 90 90 Total weight164.31 166.65 167.34 167.58 169.01 170.39

TABLE 66 Foam formulations. Eq. Polyol weight 6 7 8 9 10 11 JeffolG31-28 1951.54 77.6 48.5 48.5 48.5 48.5 48.5 Ex. 57 HSAO 386.9 0 48.5 00 48.5 0 Ex. 59 HOAO 313.4 0 0 48.5 0 0 0 Ex. 61 Soybean 293.7 19.4 0 048.5 0 48.5 % Replacement 20 50 50 50 50 50 Water 9 3.6 3.6 3.6 3.6 3.63.6 Lumulse POE 26 416.2 3 3 3 3 3 3 Tegostab B 1335.7 1 1 1 1 1 1 4690Dabco 33LV 105 0.8 0.8 0.8 0.8 0.8 0.8 Diethanolamine 35.04 1 1 1 1 1 1Niax A-1 233.7 0.1 0.1 0.1 0.1 0.1 0.1 Isocyanate 130 64.37 69.57 73.0174.22 73.29 77.94 Mondur 1488 NCO Index 90 90 90 90 90 90 Total weight170.87 176.07 179.51 180.72 180.79 185.44

The foams for tested for various mechanical properties in Examples65-71. In the Tables of these Examples, “Replacement” refers to thepercent replacement by weight of the soy-based polyether polyol withpolyols derived from algal oils.

Example 65 Density

Polyurethane foams of Example 64 were tested to measure densityaccording to standard test methods for Flexible Cellular Material-Slab,Bonded and Molded Urethane Foams (ASTM D3574). Results are shown inTable 67 and FIG. 25.

TABLE 67 Density of polyurethane foams (lbs/ft³). Replacement ControlHSAO HOAO Soy  0% 2.42 10% 2.27 2.33 2.29 20% 2.15 2.13 2.17 50% 2.232.24

Example 66 Resilience Via Ball Rebound

Polyurethane foams of Example 64 were tested to measure surfaceresilience according to standard test methods for Flexible CellularMaterial-Slab, Bonded and Molded Urethane Foams (ASTM D3574). In thistest a steel ball is dropped on the foam and rebound height is measuredas a percentage of the drop height. Results are shown in Table 68 andFIG. 26.

TABLE 68 Percent rebound of polyurethane foams. Replacement Control HSAOHOAO Soy  0% 64.17% 10% 48.78% 46.61% 47.83% 20% 36.94% 33.83% 35.22%50% 22.11% 18.33%

Example 67 Tensile Strength at Break

Polyurethane foams of Example 64 were tested to measure tensile strengthaccording to standard test methods for Flexible Cellular Material-Slab,Bonded and Molded Urethane Foams (ASTM D3574). The test measures theforce required to stretch the foams to breaking point. Results are shownin Table 69 and FIG. 27.

TABLE 69 Tensile strength of polyurethande foams - stress (kPa)Replacement Control HSAO HOAO Soy  0% 94.30 10% 106.12 102.15 106.55 20%101.60 112.24 117.93 50% 136.52 134.89

Example 68 Elongation

Polyurethane foams of Example 64 were tested to measure the percentelongation (percent that a sample will stretch) before breakageaccording to standard test methods for Flexible Cellular Material-Slab,Bonded and Molded Urethane Foams (ASTM D3574). Results are shown inTable 70 and FIG. 64.

TABLE 70 Percent elongation of polyurethane foams. Replacement ControlHSAO HOAO Soy  0% 141% 10% 142% 141% 133% 20% 139% 131% 121% 50% 115% 90%

Example 69 Compression Force Deflection

Polyurethane foams of Example 64 were tested to measure compressionforce deflection (resistance to compression) according to standard testmethods for Flexible Cellular Material-Slab, Bonded and Molded UrethaneFoams (ASTM D3574). Results are shown in Table 71 and FIG. 29.

TABLE 71 Compression force (kPa) deflection of polyurethane foams.Replacement Control HSAO HOAO Soy  0% 4.20 10% 4.60 4.38 5.15 20% 4.105.22 8.63 50% 21.41

Example 70 Compression Set

Polyurethane foams of Example 64 were tested to measure compression setaccording to standard test methods for Flexible Cellular Material-Slab,Bonded and Molded Urethane Foams (ASTM D3574). The compression set is apermanent, unrecovered loss in thickness following compression and isexpressed as a percentage of the original, uncompressed thickness.Results are shown in Table 72 and FIG. 30.

TABLE 72 Percent compression set of polyurethane foams. ReplacmentControl HSAO HOAO Soy  0% 10% 10% 10% 13% 12% 20% 14% 29% 36% 50% 47%

The loss in thickness after compression for foam derived from highstability algal polyol was surprisingly found to be 15% better than forfoam derived from high oleic algal oil and 22% better than foam derivedfrom soy at the 20% loading level.

Example 71 Hysteresis

Polyurethane foams of Example 64 were tested to measure hysteresisaccording to standard test methods for Flexible Cellular Material-Slab,Bonded and Molded Urethane Foams (ASTM D3574). Hysteresis provides ameasure of the durability of the foam to maintain its load bearingcapability, with lower percentage values reflecting lower losses infirmness is preferable. Results are shown in Table 73 and FIG. 31.

TABLE 73 Percent hysteresis of polyurethane foams. Control HSAO HOAO Soy 0% 35% 10% 48% 51% 54% 20% 58% 66% 70% 50% 95%

The described embodiments of the invention are intended to be merelyexemplary and numerous variations and modifications will be apparent tothose skilled in the art. All such variations and modifications areintended to be within the scope of the present invention. For example,the various triglyceride oils can be tailored in for a mixture ofmidchain and long chain fatty acids in order to adjust parameters suchas polarity, solvency, and foam-height of the oils or chemicals madefrom the oils. In addition, where a knockout of a gene is called for, anequivalent result may be reached using knockdown techniques includingmutation and expression of inhibitory substances such as RNAi orantisense.

1-38. (canceled)
 39. A solvent comprising an oleic acid ester preparedfrom a microalgal oil having a fatty acid profile of at least 70% oleicacids and less than 1% polyunsaturated acids.
 40. The solvent of claim39 wherein the ester is a methyl, ethyl, butyl, tert-butyl, propyl,iso-propyl, or an (ethyl)hexyl ester.
 41. The solvent of claim 39,wherein the microalgal oil has a fatty acid profile of at least 75%,80%, or 85% oleic fatty acids.
 42. The solvent of claim 39, wherein themicroalgal oil has less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%,0.2%, 0.1%, 0.05%, or 0.01% polyunsaturated fatty acids.
 43. The solventof claim 39 having a VOC (volatile organic compound) as measured by EPAmethod 24 of less than 20%, a Kauri Butanol value as measured by ASTMD1133 of greater than 50%, a kinematic viscosity at 40° C. of less than5 centistokes, and a flash point of greater than 100° C.
 44. A cleaningformulation comprising a solvent of claim 39 and an emulsifier, anon-ionic surfactant, and water.
 45. The cleaning formulation of claim44 wherein the emulsifier is Videt ME
 80. 46. The cleaning formulationof claim 45 wherein the non-ionic surfactant is Stephan BioSoft N91-8.47. The cleaning formulation of claim 46 wherein the water is deionized.48. The cleaning formulation of claim 44 further comprising limonene,methyl soyate, or methyl ricinoleate.
 49. The solvent of claim 39,wherein the oil comprises one or more of: at least 10% ergosterol;ergosterol and β-sitosterol, wherein the ratio of ergosterol toβ-sitosterol is greater than 25:1; ergosterol and brassicasterol;ergosterol, brassicasterol, and poriferasterol; and wherein the oil isoptionally free from one or more of β-sitosterol, campesterol, andstigmasterol. 50-122. (canceled)